Elimination of colonic bacterial driving lethal inflammatory cardiomyopathy

ABSTRACT

The invention relates to methods, kits and compositions for reducing the level of or eliminating Bacteroides in situ. The invention encompasses methods of preventing myocarditis, treating myocarditis or dilated cardiomyopathy, or limiting progression of myocarditis toward dilated cardiomyopathy in a subject in need thereof, comprising reducing the amount of Bacteroides sp. in the subject. The invention further encompasses methods of diagnosis of a subject as having myocarditis or dilated cardiomyopathy. The invention also encompasses compositions preventing myocarditis, treating myocarditis or dilated cardiomyopathy, or limiting progression of myocarditis toward dilated cardiomyopathy in a subject in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/225,854, filed Apr. 8, 2021, which claims the benefit of U.S.application 63/007,197 filed Apr. 8, 2020, which are herein incorporatedby reference in its entirety.

The contents of the electronic sequence listing(EB2020-03USDiv_SequenceListing.xml; Size: 26,978 bytes; and Date ofCreation: Jan. 24, 2023) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

The human microbiota comprises bacteria, archaea, viruses, and microbialeukaryotes living in our bodies. The taxonomic composition of thesecommunities has been extensively studied and is significantly associatedwith a variety of diseases and traits. This microbiota indeed consistsof thousands of different bacterial species that carry hundreds ofbillions of genes, which is called the microbiome. This microbiomeencodes for a variety of molecules (proteins, lipids, sugars, RNA, etc.)and functions that are essential and beneficial for their host, forinstance the enrichment of glycans metabolism, amino acids andxenobiotics but also the regulation of our immune system. It is alsoresponsible for the synthesis of vitamins, isoprenoids and othernutrients which results in human overall metabolism representing anamalgamation of microbial and human attributes.

Interestingly, there is a growing body of recent studies based onmetagenome sequencing that demonstrate that the presence of specificstrains (and therefore of specific genetic signatures in the microbiome)can be directly linked to a number of pathologies, includingauto-immunity, infections, inflammation or tumorigenesis.

Myocarditis is an inflammatory heart disease that develops into lethalinflammatory cardiomyopathy in 20-30% of the patients (1, 2).Myocarditis can develop into inflammatory cardiomyopathy through chronicstimulation of myosin heavy chain 6-specific Th1 and Th17 cells.However, the mechanisms that govern the cardiotoxicity programming ofheart-specific T cells have remained elusive. It is well establishedthat acute immune activation after infectious myocarditis is associatedwith the generation of autoimmune responses against myosin heavy chain 6(MYH6) (3-5), while subsequent chronic stimulation of MYH6-specific Th1and Th17 cells precipitates inflammatory cardiomyopathy (6-9).Nevertheless, it is still unclear which mechanisms mediate the initialactivation and cardiotoxicity programming of heart-specific T cells.Likewise, therapeutic approaches that mitigate the activity of suchpathogenic T cells and prevent the severe consequences of inflammatorycardiomyopathy are still limited (10, 11).

A cardinal challenge in deciphering the progressive nature of autoimmuneand chronic inflammatory diseases is the deconvolution of theirmultifactorial nature, which is determined by different degrees ofgenetic susceptibility and a multitude of environmental conditions (12,13). The quest for genetic determinants underlying susceptibility tomyocarditis and dilated cardiomyopathy (DCM) has revealed associationswith HLA-DQB1* polymorphisms (14, 15). Moreover, the development ofprogressive myocarditis in transgenic mice expressing the HLA-DQ8haplotype (encoded by the DQA1*03:01/DQB1*03:02 alleles) (7, 16)indicates that antigen presentation via certain MHC class II moleculesis a major determinant of myocarditis.

A variety of pathogens including enteroviruses, Borrelia burgdorferi andTrypanosoma cruzi can cause cardiac damage leading to myocarditisthereby predisposing affected patients for inflammatory cardiomyopathy(11). Still, the question whether and to which extent pathogen-induceddeath of cardiomyocytes results in the excessive presentation ofself-antigens to MHC class II-restricted T cells or whether antigenicmimicry of microbial components drives the disease has remainedunanswered (10, 11). The present invention fulfills this need.

BRIEF SUMMARY OF INVENTION

The invention relates to methods, kits and compositions for reducing thelevel of or eliminating a Bacteroides bacteria in situ. Preferably, theBacteroides are killed.

The invention encompasses methods of preventing myocarditis, treatingmyocarditis or dilated cardiomyopathy (DCM), or limiting progression ofmyocarditis toward DCM in a subject in need thereof, comprising reducingthe amount of Bacteroides sp. in the subject.

The invention encompasses methods of diagnosis of a subject as havingmyocarditis or DCM, comprising obtaining a biological sample of thesubject, quantifying the amount of Bacteroides sp. in the biologicalsample relative to a control sample.

The invention also encompasses methods of treating myocarditis or DCM ina subject, comprising obtaining a biological sample of the subject,quantifying the amount of Bacteroides sp. in the biological samplerelative to a control sample, and when the amount of Bacteroides sp. ishigher in the biological sample relative to a control sample, reducingthe amount of Bacteroides sp. in the subject.

The invention encompasses compositions preventing myocarditis, treatingmyocarditis or DCM, or limiting progression of myocarditis toward DCM ina subject in need thereof. Preferably, the compositions kill Bacteroidesbacteria. Alternatively, the compositions may reduce the amount ofBacteroides sp. in the subject without killing Bacteroides bacteria.

In various embodiments, the method comprises administering to thesubject an effective amount of an antibiotic, bacteria, engineeredbacteria, phage, recombinant phage, packaged phagemid, wild-type orsynthetic endolysin, wild-type or synthetic bacteriocin or anycombination thereof.

In various embodiments, the composition comprises an effective amount ofan antibiotic, engineered bacteria, phage, recombinant phage, packagedphagemid, endolysin, bacteriocin or any combination thereof.

In various embodiments, the antibiotic is selected from streptomycin,vancomycin, clindamycin, metronidazole, sulphadoxine, trimethoprim, orany combination of 1, 2, 3, 4, 5 or 6 of these antibiotics.

In various embodiments, the phage, recombinant phage or packagedphagemid encodes an endolysin.

In various embodiments, the phage, recombinant phage, packaged phagemidencodes a nuclease selected from CRISPR-Cas, TALENs and variants, zincfinger nuclease (ZFN) and ZFN variants, natural, evolved or engineeredmeganuclease or recombinase variants.

In various embodiments, the bacteria or engineered bacteria does notproduce MYH6 mimic peptides (in particular mimics of MYH6₆₁₄₋₆₂₉ orMYH6₆₁₄₋₆₂₈ peptides, typically of sequence SEQ ID NO: 10, 16 or 17),more particularly which does not produce β-gal₁₁₋₂₅ peptide (typicallyof sequence SEQ ID NO: 11, 12, 18, 19, 20, 21 or 22), still particularlydoes not produce a β-galactosidase.

In various embodiments, the Bacteroides is B. thetaiotaomicron and/or B.faecis. Preferably, the Bacteroides is B. thetaiotaomicron and/or B.faecis which produce MYH6 mimic peptides (in particular mimics ofMYH6₆₁₄₋₆₂₉ or MYH6₆₁₄₋₆₂₈ peptides, typically of sequence SEQ ID NO:10, 16 or 17), more particularly which produce β-gal₁₁₋₂₅ peptide(typically of sequence SEQ ID NO: 11, 12, 18, 19, 20, 21 or 22).

The invention encompasses the following embodiments:

A method of preventing myocarditis in a subject in need thereof,comprising reducing the amount of Bacteroides sp. in a subject.

A method of treating myocarditis or DCM in a subject in need thereof,comprising reducing the amount of Bacteroides sp. in a subject.

A method of limiting progression of myocarditis toward DCM in a subjectin need thereof, comprising reducing the amount of Bacteroides sp. in asubject.

A method of diagnosis a subject as having myocarditis or DCM, comprisingobtaining a biological sample of the subject, quantifying the amount ofBacteroides sp. in the biological sample relative to a control sample.

Any of these methods, comprising administering to the subject aneffective amount of an antibiotic, phage, recombinant phage, packagedphagemid, bacteria, engineered bacteria, bacteriocin or endolysin. Insome embodiments, the antibiotic is selected from streptomycin,vancomycin, clindamycin, metronidazole, sulphadoxine, trimethoprim, orany combination of 1, 2, 3, 4, 5 or 6 of these antibiotics. In someembodiments, the phage, recombinant phage or packaged phagemid encodes anuclease selected from CRISPR-Cas, TALENs and variants, zinc fingernuclease (ZFN) and ZFN variants, natural, evolved or engineeredmeganuclease or recombinase variants. In some embodiments, theBacteroides is B. thetaiotaomicron and/or B. faecis.

A composition for preventing myocarditis in a subject in need thereof,comprising a pharmaceutical agent which reduces the amount ofBacteroides sp. in a subject.

A composition for treating myocarditis or DCM in a subject in needthereof, comprising a therapeutic agent which reduces the amount ofBacteroides sp. in a subject.

A composition for limiting progression of myocarditis toward DCM in asubject in need thereof, comprising a pharmaceutical agent which reducesthe amount of Bacteroides sp. in a subject.

Any of these compositions, comprising an effective amount of anantibiotic, phage, recombinant phage, packaged phagemid, bacteria,engineered bacteria, bacteriocin or endolysin. In some embodiments, theantibiotic is selected from streptomycin, vancomycin, clindamycin,metronidazole, sulphadoxine, trimethoprim, or any combination of 1, 2,3, 4, 5 or 6 of these antibiotics. In some embodiments, the phage,recombinant phage or packaged phagemid encodes a nuclease selected fromCRISPR-Cas, TALENs and variants, zinc finger nuclease (ZFN) and ZFNvariants, natural, evolved or engineered meganuclease or recombinasevariants. In some embodiments, the Bacteroides is B. thetaiotaomicronand/or B. faecis.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-R. Microbiome-dependent transition of autoimmune myocarditis todilated cardiomyopathy. (FIG. 1A) Survival of TCRM mice under SPF or GFconditions. (FIG. 1B) Gross pathology of hearts from 12 week old SPFTCRM and age-matched GF TCRM mice. (FIG. 1C) Histological analysis ofhearts of 12 week old TCRM mice kept under SPF or GF conditions usinghematoxylin-eosin (HE) and elastica-van Gieson (EVG) staining. (FIG. 1D)Histopathological disease severity in TCRM mice under SPF and GFconditions. Dots represent values of individual mice; bar indicates meandisease severity. (FIG. 1E-G) Echocardiographic parameters in TCRM micekept under SPF or GF conditions and in transgene-negative littermate ofcontrols with (FIG. 1E) ejection fraction, (FIG. 1F) fractionalshortening and (FIG. 1G) systolic left ventricular internal diameter(LVID) determined in individual mice. (FIG. 1H-J) Co-housing of GF TCRMmice with SPF TCRM mice at the age of 4 and 8 weeks. (FIG. 1H) Schematicrepresentation of co-housing experiments. (FIG. 1I) Prospective survivalanalysis of TCRM mice, arrowheads indicate the age of transfer to SPFconditions. (FIG. 1J) Development of myocarditis in TCRM mice under SPF,GF and co-housing conditions; dots represent disease severity inindividual mice; bar indicates mean disease severity. (FIG. 1K and L)Enumeration of heart-infiltrating cells of 12 week old TCRM mice underSPF and GF conditions or GF TCRM mice co-housed (CoH) for 4 weeks in SPFconditions with (FIG. 1K) CD45⁺ cells and (FIG. 1L) myosin-specific(Vβ8⁺CD4⁺) cells. (FIG. 1M) IFN-γ- and (FIG. 1N) IL-17-producingheart-infiltrating MYH6-specific CD4⁺ T cells (Vβ8⁺CD4⁺). (FIG. 1O-R)Heart-infiltrating myeloid cell subsets from SPF, GF or 4 week cohousedTCRM mice analyzed by flow cytometry; dots represent values fromindividual mice, line indicates mean value. (FIG. 1O) RepresentativetSNE plots of myeloid cells in the heart of TCRM SPF mice. Enumerationof (FIG. 1P) inflammatory monocytes, (FIG. 1Q) MHCII^(high) macrophagesand (FIG. 1R) MERTK⁺ macrophages in the heart (mean±SEM). Pooled datafrom 2 independent experiments n=5-6 mice (FIG. 1K, L, P-R) or threeindependent experiments with ≥15 mice (FIG. 1A and I), ≥12 mice (FIG.1B, C and D), ≥6 mice (FIG. 1E-G), ≥11 mice (FIG. 1J)≥7 mice (FIG. 1M)per group. Representative micrographs of hearts and heart sections fromone out of at least 12 mice (FIG. 1A, B). Statistical analysis wasperformed using Student's t test (FIG. 1D) or one-way ANOVA withDunnett's multiple comparison test (FIG. 1E-G, J-N and P-R) with *,p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 2A-M. Interaction of MYH6-specific CD4⁺ T cells with theintestinal microbiome. (FIG. 2A) Flow cytometric analysis of mucosalhoming markers in heart-infiltrating MYH6 specific cells from SPF or GFTCRM mice. (FIG. 2B-C) Location and proliferation of CFSE-labelled TCRMcells after adoptive transfer to Rag1^(−/−) mice. (FIG. 2B) Confocalmicroscopy analysis of colonic patches at day 3 post adoptive transfer,scale bar 100 μm (FIG. 2C) Proliferation of MYH6-specific cells flowcytometry-based quantification of CFSE dilution in the indicated organsand at the indicated time points (mean±SEM). (FIG. 2D) Microbiomeanalysis of 12 week old transgene-negative littermate controls (Tg⁻),SPF TCRM and GF TCRM mice co-housed at 4 weeks of age under SPFconditions. (FIG. 2E) Principal component analysis of the fecalbacterial composition. (FIG. 2F) Heat map of the relative abundance ofthe indicated bacterial classes and families in feces. (FIG. 2G-H)MYH6-specific CD4⁺ T cell crossreactivity. (FIG. 2G) In vitroproliferation of CD4⁺ T cells from TCRM mice after re-stimulation withMYH6, gal peptide from Bacteroides and cysteine hydrolase-derivedpeptide from Enterobacter determined by CFSE dilution assay. (FIG. 2H)Cytokine production of heart- and colon-infiltrating, Vβ8-expressingCD4⁺ T cells from TCRM mice under SPF conditions after ex vivore-stimulation with MYH6 peptide and Bacteroides β-gal peptide (box andwhiskers show mean±interquartile range). (FIG. 2I-M) GF TCRM mice weremonocolonized with parental B. thetaiotaomicron (wild type, WT) or B.thetaiotaomicron lacking the β-galactosidase BT1626 (B. thetaiotaomicronΔβ-gal). (FIG. 2I) Schematic representation of the experimental setting.Flow cytometric analysis of (FIG. 2J) heart-infiltrating cells and (FIG.2K) MYH6-specific cytokine producing cells in the heart and colon (boxand whiskers show mean±interquartile range). (FIG. 2L) IgA bound fecalbacteria determined by bacterial flow cytometry and (FIG. 2M) pooleddata shown as mean±SEM. Pooled data from n=6 (FIG. 2A-F, J-M), n=7 (FIG.2G) and n=5 (FIG. 2H) mice from at least two independent experiments.Representative histograms from two independent experiments withduplicates (FIG. 2G). Statistical analysis was performed using Student'st test (FIG. 2A, H, J, K, M) or one-way ANOVA with Dunnett's multiplecomparison test (FIG. 2F) with *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 3A-K. Impact of antibiotics treatment on lethal heart disease andimmune reactivity in the TCRM model. (FIG. 3A) Survival, (FIG. 3B)disease severity, and (FIG. 3C) histological analysis of hearts of TCRMmice after post-weaning oral treatment with a broad spectrum antibioticscombination comprising of Sulphadoxine, Trimethoprim and Metronidazole(S+T+M); disease severity was determined in 20 week old mice; dotsrepresent values of individual mice; bar indicates mean diseaseseverity. (FIG. 3D-J) Effect of antibiotics treatment regimen onmyocarditis progression in the adoptive transfer myocarditis model.(FIG. 3D) Schematic representation of the experimental set up. (FIG. 3E)B. thetaiotaomicron quantification in feces of mice by qPCR in theindicated treatment groups. (FIG. 3F) Histopathological analysis ofhearts from Rag1^(−/−) mice treated with antibiotics at day 28; dotsrepresent values of individual mice; bar indicates mean diseaseseverity. Quantification of heart-infiltrating CD45⁺ cells (FIG. 3G) andVα8-expressing CD4⁺ T cells (FIG. 3H) in Rag1^(−/−) mice at day 28.(FIG. 3I) Cytokine production of heart-infiltrating MYH6-specificVβ8⁺CD4⁺ T cells (box and whiskers show mean±interquartile range). (FIG.3J) Heat map representation of the specific IgG responses against B.thetaiotaomicron, B distasonis, B. vulgatus and E. cloacae determined byELISA. (FIG. 3K) B. thetaiotaomicron-specific IgG response in sera ofBALB/c mice or BALB/c mice adoptively transferred with CD4⁺ TCRM T cells(box and whiskers show mean±interquartile range). Pooled data from twoto three independent experiments with n=7-10 (FIG. 3A-C), n=5-13 mice(FIG. 3G-J) and n=5-7 (FIG. 3K). Representative sections from one out of10 or 7 mice (FIG. 3C). Statistical analysis was performed usingStudent's t test (FIG. 3K) or Mann-Whitney test (FIG. 3B); one-way ANOVAwith Dunnett's multiple comparison test (FIG. 3E-I) with *, p<0.05; **,p<0.01; ***, p<0.001.

FIGS. 4A-M. Immune reactivity against Bacteroides and cardiac myosinantigens in human myocarditis patients. (FIG. 4A) Analysis of B.thetaiotaomicron-specific IgG antibodies in sera of myocarditis patientsfrom the AMITIS cohort at first admission and different time pointspost-diagnosis compared to sera from a cohort of healthy individuals,light grey and dark grey squares indicate patients with high or lowanti-B. thetaiotaomicron antibody levels respectively, cut value wasdetermined as the mean+2SD of the healthy group; dots representindividual antibody levels, bar indicates mean value. (FIG. 4B) Ejectionfraction (EF) and (FIG. 4C) C-reactive protein (CRP) values in patientsfrom the AMITIS cohort at the indicated time points; individual valuesare shown, bar represents mean value. (FIG. 4D) Composite clinicalscores of myocarditis patients from the AMITIS cohort with low vs highIgG antibodies against B. thetaiotaomicron at the first visit (asindicated in A); dots represent individual clinical scores as the sum ofpositivity for anti-beta1-AR antibodies, CRP values and proportion of EF(FIG. 4E-F) Anti-bacterial IgG antibody responses in sera of myocarditispatients and healthy controls. (FIG. 4E) Heat map representation ofspecific IgG response against B. thetaiotaomicron, B. distasonis, B.vulgatus and E. cloacae in the AMITIS cohort. (FIG. 4F) B.thetaiotaomicron-specific IgG in serum of patients of the Micro-DCMcohort at admission (individual values are shown, bar indicates meanvalue) and (FIG. 4G) heat map representation of antibacterial IgGreactivity in the Micro-DCM cohort. (FIG. 4H) Heat maps representing thebinding of the MYH6₆₁₄₋₆₂₉ peptide or the B. thetaiotaomicron β-gal₁₁₋₂₅peptide. The in-silico analysis was done for molecules that cover the99% of the HLA-DQ alleles in open population. (FIG. 4I) IFN-γ ELISPOTanalysis of peripheral blood mononuclear cells of HLA-DQB1*03 healthyvolunteers (n=10) or patients from the Micro-DCM cohort (HLA-DQB1*03n=9; other HLA n=8) after stimulation with the human-MYH6₆₁₄₋₆₂₉ peptideor the B. thetaiotaomicron β-gal₁₁₋₂₅ peptides (violin plots showmean±interquartile range). (FIG. 4J) Schematic representation of fecaltransplant experiment using stool from two myocarditis patients(HLA-DQB1*03:02 haplotype and B. thetaiotaomicron 16S rRNA-positive) toGF TCRM or transgene-negative (Tg⁻) control mice. (FIG. 4K) B.thetaiotaomicron quantification in feces of TCRM or Tg⁻ controls at theindicated times post fecal transplantation (box and whiskers showmean±interquartile range). (FIG. 4L-M) Enumeration of heart-infiltratingCD45⁺ immune (FIG. 4L) and CD4⁺ T cells (FIG. 4M) in recipient mice at 4weeks after fecal transplantation (mean±SEM). Statistical analysis wasperformed using one-way ANOVA with Dunnett's multiple comparison test(FIG. 4A-C and I) or Mann-Whitney U test (FIG. 4D) or Student's t test(FIG. 4E-D and (L-M) with *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 5A-F. Impact of microbial colonization on heart function andinflammation in TCRM mice. (FIG. 5A-C) Echocardiographic parameters inTCRM mice under SPF, GF and co-housing conditions with (FIG. 5A)ejection fraction, (FIG. 5B) systolic left ventricular internal diameter(LVID) and (FIG. 5C) fractional shortening determined in individualmice. (FIG. 5D-F) Flow cytometric analysis of heart-infiltrating myeloidcells. Representative dot plots showing CCR2 and Ly6C (FIG. 5D) and CD64and MHCII expression (FIG. 5E) shown as percentage of CD11b⁺ Ly6G⁻ cellsdepicted for each condition. (FIG. 5F) Enumeration of differentsubpopulations of myeloid cells in TCRM mice kept under the indicatedconditions (mean values±SEM; n=5-8 mice per group). Statistical analysiswas performed using one-way ANOVA with Dunnett's multiple comparisontest with *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 6A-1 . Activation TCRM CD4⁺ cells and interaction with theintestinal microbiome. (FIG. 6A) Schematic representation of theexperimental set up for the analysis of homing and early proliferationof CFSE-labelled TCRM cells in Rag^(−/−) mice. (FIG. 6B-C) Histologicalanalysis of colonic (FIG. 6B) and mesenteric (FIG. 6C) lymph nodes ofRae mice 3 days after a.t. of CFSE labelled TCRM cells, scale bar=30 μm.(FIG. 6D) Flow cytometric analysis of the proliferation patterns of TCRMcells in the colonic lamina propria and heart at the indicated timepoints after a.t. to Rae mice; percentages indicate CFSE^(low/neg)MYH6-specific cells in the indicated organs. (FIG. 6E) Cytokine profilesof heart-infiltrating and colonic Vβ8-expressing CD4⁺ T cells from TCRMmice at the indicated time points analyzed by flow cytometry (mean±SEM).(FIG. 6F) Flow cytometry-based cytokine profile analysis ofVβ8-expressing CD4⁺ T cells from the colonic lamina propria of TCRM miceor transgene-negative littermates (Tg⁻). (FIG. 6G) Production of IFN-©and IL-17 by colonic CD4⁺ T cells from TCRM mice or DO11.10 mice afterex-vivo re-stimulation with MYH6₆₁₄₋₆₂₉; representative dot-plots areshown. (FIG. 6H) Bacterial α-diversity in feces of 12 week old TCRM miceand transgene-negative littermates (Tg−) under SPF conditions. (FIG. 6I)Relative abundance of Bacteroides and Parabacteroides in fecal samplesof 12 weeks of age TCRM or Tg⁻ mice under SPF or cohousing conditions(box and whiskers show mean±interquartile range). (n=5-13 mice per groupfrom 2-3 independent experiments). Statistical analysis was performedusing Student's t test or one-way ANOVA with Dunnett's multiplecomparison test with *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 7A-I. Specificity and immune interaction of Bacteroides mimicpeptide-specific CD4⁺ T cells. (FIG. 7A) In vitro proliferation of CD4⁺T cells from TCRM mice or DO11.10 mice measured by CFSE dilution afterre-stimulation with MYH6 or Bacteroides β-galactosidase peptide orovalbumin peptide at indicated concentration (representative histogramsfrom 1 out of 2 experiments) (FIG. 7B) Dendritic cells loaded with MYH6or β-gal peptide from Bacteroides at the indicated concentration wereco-cultured in vitro with CD4⁺ T cells from TCRM mice. Maximumproliferation of CD4⁺ T cells was analyzed by flow cytometry andeffective concentration 50 (EC₅₀) for each peptide was determined. (FIG.7C) CD4⁺ T cells from TCRM mice were adoptively transferred into BALB/cmice. Proliferation of CD4⁺ T cells from TCRM mice was determined exvivo by CFSE dilution after injection of peptide loaded dendritic cells(n=3). (FIG. 7D-FIG. 7E) Colonization of GF TCRM mice with SPFmicrobiota or B. thetaiotaomicron or E. coli. Induction of IFN-© andIL-17 in Vβ8-expressing CD4⁺ T cells in the colonic lamina propria ofTCRM mice at 8 weeks after bacterial colonization. Fold increase valueswere calculated using the cytokine production in GF mice as baseline(mean±SEM). (FIG. 7F) Strategy for the generation of the β-galactosidaseBT1626 deficient B. thetaiotaomicron strain. (FIG. 7G) Colonizationefficiency of WT the parental B. thetaiotaomicron (ATCC29148^(Δtdk)) andthe Δβ-gal B. thetaiotaomicron (ATCC29148^(Δtdk) lacking theBT1626β-galactosidase) in GF BALB/c mice (mean±SEM). (FIG. 7H-I) Bacterialflow of fecal IgA bound bacteria in TCRM and Tg⁻ 12 weeks old mice.(FIG. 7H) experimental dosing, (FIG. 7I) representative dot plots leftand quantification right of IgA bound bacteria; background was set usingsamples from Rag^(−/−) mice (mean±SEM). (n=3-11 mice per group from 2-3independent experiments). Statistical analysis was performed usingStudent's t test or one-way ANOVA with Dunnett's multiple comparisontest with *, p<0.05; **, p<0.01; ***, p<0.001

FIGS. 8A-F. Effect of antibiotics treatment on splenic and colonic CD4⁺T cells. (FIG. 8A) Microbiota composition analysis by 16S rRNAsequencing of mice treated with a broad spectrum antibiotics cocktailcontaining sulphadoxine, trimethoprim and metronidazole (S+T+M) comparedto untreated TCRM mice. (FIG. 8B-F) Rag1^(−/−) mice were adoptivelytransferred with 10⁶ splenocytes from TCRM mice and treated with theindicated antibiotics. Flow cytometry-based enumeration of colonic (FIG.8B) CD45⁺ and (FIG. 8C) Vβ8-expressing CD4⁺ T and (FIG. 8D)Vβ8-expressing CD4⁺ T cells in spleens. (FIG. 8E) Cytokine production ofheart infiltrating Vβ8-expressing CD4⁺ T cells with representative dotplots. (FIG. 8F) IFN-© and IL-17 production in colonic Vβ8-expressingCD4⁺ T cells. Box and whiskers show mean±interquartile range; n=6-8 micefrom 2 or 3 independent experiments. Statistical analysis was performedusing one-way ANOVA with Dunnett's multiple comparison test with *,p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 9A-L. B and T cell responses in myocarditis patients. (FIG. 9A-C)Clinical parameters at admission in B. theta high or low AMITISpatients. Antibody responses in patients from the AMITIS (FIG. 9D-F) andMicro-DCM (FIG. 9G-J) cohorts compared to healthy individuals. IgGantibodies against E. cloacae (FIG. 9D and FIG. 9G), E. coli (FIG. 9Eand FIG. 9H), B. distasonis (FIG. 9I) and B. vulgatus (FIG. 9J) weredetermined by ELISA and total IgG concentrations (FIG. 9F) weredetermined by turbidimetry; dots represent individual patients, barindicates mean values. (FIG. 9K) Correlation between MYH6- or β-galIFN-γ-producing cells in myocarditis patients of the Micro-DCM cohortand healthy individuals with the indicated HLA-DQB1 alleles; dotsrepresent individual values. (FIG. 9L) Graphical representation of themultiple risk factors that regulate the outcome of inflammatorycardiomyopathy. Statistical analysis was performed using Student's ttest (FIG. 9G-J) or one-way ANOVA with Dunnett's multiple comparisontest with (FIG. 9D-F) or Pearson correlation coefficient with two tailedP-value calculation.*, p<0.05.

DETAILED DESCRIPTION OF INVENTION

The present invention unveils the hitherto unknown connection betweenthe microbiome and inflammatory cardiac disease in humans. The inventorsshow here that commensal bacteria such as Bacteroides can serve as asource of a mimic peptide that promotes the progression of myocarditisto inflammatory cardiomyopathy. The presented invention also shows thatreducing the level of or eliminating Bacteroides in situ with treatmentsuch as antibiotics can prevent lethal heart failure.

In at least a substantial proportion of myocarditis patients, thepresence of specific bacteria in the intestinal microbiome that produceMYH6 mimic peptides and the genetic background with the particular setof HLADQB1*03 alleles determine the risk for the development ofinflammatory myocarditis (FIG. 9L). In this scenario, cross-reactiveCD4⁺ T cells primed in intestinal microenvironments can enter themyocardium and exacerbate the damage caused by noxious agents orprocesses such as infection by cardiotropic viruses or subclinicalmyocardial infarction (FIG. 9L). Likewise, unleashing control over self-and cross-reactive T cells during immune checkpoint inhibitor therapymight be a reason for potentially lethal cardiac inflammation (30).Indeed, fulminant myocarditis during combination immune checkpointblockade was found to affect patients who shared the HLADQB1*03:01allele (31). Thus, the invention provides the rationale for extendedprospective clinical trials to further dissect the connection betweenthe microbiome-driven activation of cross-reactive CD4+ T cells,HLA-dependent genetic predisposition and inflammatory cardiomyopathy.Targeting of the microbiome of genetically predisposed myocarditispatients or susceptible patients undergoing checkpoint inhibitortreatment through antibiotics may alleviate disease severity and cantherefore contribute to the prevention of the potentially lethalsequelae of inflammatory cardiomyopathy.

To treat microbiome-associated diseases or disorders, unwantedBacteroides, typically unwanted Bacteroides producing MYH6 mimicpeptides, can be eliminated by using subtractive methods such asantibiotics, phages, recombinant phages, packaged phagemids, wild-typeof synthetic endolysins, wild-type of synthetic bacteriocins (such ascolicins, pyocins and/or tailocins) which result in the killing ofdeleterious Bacteroides bacteria or by using competitive methods such asbacteria or engineered bacteria which do not produce said MYH6 mimicpeptides and preferably present a competitive advantage over unwantedBacteroides, which results in the replacement of deleterious Bacteroidesbacteria by said non-deleterious bacteria or engineered bacteria. Suchstrategies can significantly reduce the load of Bacteroides populations.

The invention encompasses compositions, kits and methods for eliminatinga Bacteroides bacteria in situ. The compositions, kits and methods ofthe invention eliminate deleterious Bacteroides bacteria, in particularBacteroides bacteria producing MYH6 mimic peptides (in particular mimicsof MYH6₆₁₄₋₆₂₉ or MYH6₆₁₄₋₆₂₈ peptides, typically of sequence SEQ ID NO:10, 16 or 17), more particularly Bacteroides bacteria producingβ-gal₁₁₋₂₅ peptide (typically of sequence SEQ ID NO: 11, 12, 18, 19, 20,21 or 22), within the host microbiome by killing or reducing the growthof these Bacteroides.

The invention encompasses the use of a vector that can transfer withhigh efficiency a nucleic acid, preferably a plasmid, into a bacterialpopulation within the microbiome that allows the expression of anexogenous enzyme that will modify a gene sequence or directly kill theBacteroides bacteria. The invention further includes methods forscreening for elimination of the Bacteroides, for determining theefficiency of vectors at eliminating these Bacteroides, and fordetermining the effects of these vectors. Preferably, the elimination ofBacteroides bacteria in situ involves the use of antibiotics, ofwild-type of synthetic bacteriocins, the use of phages, recombinantphage, packaged phagemid, wild-type of synthetic endolysins, introducinga double strand break in the DNA sequence, or any combination thereof.In an embodiment, the elimination of Bacteroides bacteria in situinvolves the use of bacteria or engineered bacteria, in particularengineered Bacteroides bacteria, which do not produce MYH6 mimicpeptides (in particular mimics of MYH6₆₁₄₋₆₂₉ or MYH6₆₁₄₋₆₂₈ peptides,typically of sequence SEQ ID NO: 10, 16 or 17), more particularly whichdo not produce β-gal₁₁₋₂₅ peptide (typically of sequence SEQ ID NO: 11,12, 18, 19, 20, 21 or 22).

The invention further includes methods for pre-treatment screening ofpatient to assess the relevancy to treat a patient based on the presenceof specific bacteria in the patient intestinal microbiome that produceMYH6 mimic peptides, the genetic background of the patient with theidentification of particular set of HLADQB1*03 alleles and inflammatorymyocarditis symptoms of the patient. The invention further includes apre-treatment screening of effective antibiotics, effectivebacteriocins, effective phages, effective recombinant phages, effectivepackaged phagemids, effective endolysins, effective bacteria orengineered bacteria or any combination thereof against the targetedBacteroides bacteria.

Provided herein are compositions, kits and methods of treating,preventing or curing myocarditis or DCM by killing or reducing thegrowth of a Bacteroides sp.

The invention encompasses the use of a vector that can transfer withhigh efficiency a nucleic acid, preferably a plasmid, into a bacterialpopulation within the microbiome that allows the expression of anexogenous enzyme that will modify a gene sequence or directly kill theBacteroides bacteria.

Methods of Treatment

The invention encompasses methods of preventing myocarditis, treatingmyocarditis or DCM, or limiting progression of myocarditis toward DCM ina subject in need thereof, comprising reducing the amount of Bacteroidessp. in the subject.

The invention encompasses methods of reducing or eliminating aBacteroides sp. in situ.

In one embodiment, the Bacteroides sp. is reduced or eliminated bycontacting it with an antibiotic, for example, as described Gil-Cruz etal., Science 366, 881-886 (2019), which is hereby incorporated byreference.

In one embodiment, the Bacteroides sp. is reduced or eliminated by agenetic modification that leads to the death or reduction in growth of aBacteroides sp. bacteria, for example, as described in Bikard et al.,Cell Host Microbe, Vol. 12, 177-186 (2012) and Bikard et al., NatureBiotechnology Vol. 32 (11) 1146-51 (2014).

In one embodiment, the Bacteroides sp. is reduced or eliminated bycontacting it with an endolysin, for example, as described in Gerstmanset al., Biochem Soc Trans. 2016 February; 44(1):123-8, which is herebyincorporated by reference.

In one embodiment, the Bacteroides sp. is reduced or eliminated bycontacting it with a bacteriocin (such as a colicin, a pyocin and/or atailocin).

In one embodiment, the Bacteroides sp. is reduced or eliminated byadministering an bacteria or an engineered bacteria, in particularengineered Bacteroides sp. which do not produce MYH6 mimic peptides (inparticular mimics of MYH6₆₁₄₋₆₂₉ or MYH6₆₁₄₋₆₂₈ peptides, typically ofsequence SEQ ID NO: 10, 16 or 17), more particularly which do notproduce β-gal₁₁₋₂₅ peptide (typically of sequence SEQ ID NO: 11, 12, 18,19, 20, 21 or 22), and preferably present a competitive advantage overBacteroides sp. to be reduced or eliminated, for example which comprisean heterologous gene or gene circuit required to import and/ormetabolize a particular exogenous food source, such as an exogenoussugar.

By “engineered bacteria” is meant herein a bacteria which has beengenetically modified, compared to the wild-type strain, in particular toexpress proteins of interest and/or to stop expressing unwantedproteins.

In a particular embodiment, when said engineered bacteria comprise anheterologous gene or gene circuit required to import and/or metabolize aparticular exogenous food source, such as an exogenous sugar, saidengineered bacteria are used in combination with said exogenous foodsource.

In one embodiment, the method comprises contacting the unwanted bacteriawith an effective amount of an antibiotic, phage, recombinant phage,packaged phagemid, wild-type or synthetic bacteriocin wild-type orsynthetic endolysin or any combination thereof.

In one embodiment, the antibiotic is selected from streptomycin,vancomycin, clindamycin, metronidazole, sulphadoxine, trimethoprim, orany combination of 1, 2, 3, 4, 5 or 6 of these antibiotics.

In one embodiment, the phage, recombinant phage, packaged phagemidencodes a wild-type or synthetic endolysin and/or a wild-type orsynthetic bacteriocin

In one embodiment, the phage, recombinant phage, packaged phagemidencodes a nuclease selected from the group consisting of CRISPR-Cas andvariants, TALENs and variants, zinc finger nuclease (ZFN) and ZFNvariants, natural, evolved or engineered meganuclease or recombinasevariants.

In one embodiment, the Bacteroides is B. thetaiotaomicron or B. faecis.

In a preferred embodiment, the Bacteroides sp. are contacted in situwith a vector that can transfer with high efficiency a nucleic acid intothe bacteria to express an exogenous enzyme (such as Cas9 or Cpf1 alsoknown as Cas12a) in the bacteria that results in bacterial cell death orreduced growth.

In a preferred embodiment, the exogenous enzyme can result in a geneticmodification where Cas9 nuclease is used to make the desiredmodification. Thus, the invention contemplates introducing a doublestrand break in the DNA of the bacterial DNA at a specific sequence, forexample with a CRISPR/Cas system, together with non-homologous endjoining (NHEJ) or homologous recombination (HR) to generate the desiredgenetic modification. Preferably, the double strand break is generatedin the presence of an editing template comprising homologous region withDNA regions located around the specific sequence located in thebacterial DNA.

The genetic modification can be a point mutation(s), a deletion(s),insertion(s) or any combination thereof. Preferably, the geneticmodification is a point mutation, an insertion or a deletion inside acoding sequence leading to a frameshift mutation or a deletion mutation.The genetic modification preferably eliminates, reduces, or increasesthe expression of a gene. The genetic modification can be in thetranslated or untranslated regions of a gene. The genetic modificationcan be in the promoter region of a gene or within any other regioninvolved in gene regulation. In one embodiment, the genetic modificationintegrates a phage genome or exogenous DNA into the host bacterialchromosome or endogenous plasmid(s). In one embodiment, the geneticmodification results in expression of an exogenous protein from anintegrated exogenous DNA in the host bacterial chromosome or endogenousplasmid(s). Most preferably, the genetic modification involves eitherNHEJ or HR endogenous repair mechanism of the host bacteria.

In some embodiments, the genetic modification results in the change inat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45, 50, 100, 200, 500, etc. amino acids to a different aminoacid. In some embodiments, the genetic modification introduces a stopcodon. In some embodiments, the genetic modification is outside proteincoding sequences, within RNA, or within regulatory sequences.

In some embodiments, the genetic modification is within aβ-galactosidase gene, particularly in the coding or non-coding sequenceof the β-galactosidase gene, more particularly in the coding ornon-coding sequence of the BT-1626 gene, more particularly in the geneencoding the protein of sequence SEQ ID NO: 1, in particular in thenucleic acid sequence SEQ ID NO: 2.

Bacterial Elimination with Antibiotics

Particular bacteria or groups of bacteria such as Bacteroides sp. can beeliminated by treatment with antibiotic(s). Thus, the inventionencompasses methods of treating a subject with an antibiotic.Preferably, the level of Bacteroides sp. is measured before and afterthe treatment.

In one embodiment, the invention encompasses a method comprisingmeasuring the level of a Bacteroides sp., subsequently administering anantibiotic, and measuring the level of the Bacteroides sp. after thetreatment.

In some embodiments, the antibiotic is streptomycin, vancomycin,clindamycin, or metronidazole, alone or in any possible combination. Insome embodiments, the antibiotic is sulphadoxine, trimethoprim, ormetronidazole, alone or in any possible combination, such as describedin Gil-Cruz et al., Science 366, 881-886 (2019), which is herebyincorporated by reference. In some embodiments, the antibiotic isselected from streptomycin, vancomycin, clindamycin, metronidazole,sulphadoxine, trimethoprim, or any combination of 1, 2, 3, 4, 5 or 6 ofthese antibiotics.

In some embodiments, the antibiotic is selected from the groupconsisting of penicillins such as penicillin G, penicillin K, penicillinN, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin,oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin,pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin,epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, andpiperacillin; cephalosporins such as cefacetrile, cefadroxil,cephalexin, cephaloglycin, cefalonium, cefaloridine, cefalotin,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine,cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime,cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef, cefbuperazone,cefminox, cefotetan, cefoxitin, cefotiam, cefcapene, cefdaloxime,cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime,cefotaxime, cefovecin, cefpimizole, cefpodoxime, cefteram, ceftamere,ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone,cefoperazone, ceftazidime, latamoxef, cefclidine, cefepime, cefluprenam,cefoselis, cefozopran, cefpirome, cefquinome, flomoxef, ceftobiprole,ceftaroline, ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor,cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium,cefoxazole, cefrotil, cefsumide, ceftioxide, cefuracetime, andnitrocefin; polymyxins such as polysporin, neosporin, polymyxin B, andpolymyxin E, rifampicins such as rifampicin, rifapentine, and rifaximin;Fidaxomicin; quinolones such as cinoxacin, nalidixic acid, oxolinicacid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin,enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin,ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin,levofloxacin, pazufloxacin, temafloxacin, tosufloxacin, clinafloxacin,gatifloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin,prulifloxacin, delafloxacin, nemonoxacin, and zabofloxacin; sulfonamidessuch as sulfafurazole, sulfacetamide, sulfadiazine, sulfadimidine,sulfafurazole, sulfisomidine, sulfadoxine, sulfamethoxazole,sulfamoxole, sulfanitran, sulfadimethoxine, sulfametho-xypyridazine,sulfametoxydiazine, sulfadoxine, sulfametopyrazine, and terephtyl;macrolides such as azithromycin, clarithromycin, erythromycin,fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin,midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin,tylosin, and roxithromycin; ketolides such as telithromycin, andcethromycin; fluoroketolides such as solithromycin; lincosamides such aslincomycin, clindamycin, and pirlimycin; tetracyclines such asdemeclocycline, doxycycline, minocycline, oxytetracycline, andtetracycline; aminoglycosides such as amikacin, dibekacin, gentamicin,kanamycin, neomycin, netilmicin, sisomicin, tobramycin, paromomycin, andstreptomycin; ansamycins such as geldanamycin, herbimycin, andrifaximin; carbacephems such as loracarbef; carbapenems such asertapenem, doripenem, imipenem (or cilastatin), and meropenem;glycopeptides such as teicoplanin, vancomycin, telavancin, dalbavancin,and oritavancin; lincosamides such as clindamycin and lincomycin;lipopeptides such as daptomycin; monobactams such as aztreonam;nitrofurans such as furazolidone, and nitrofurantoin; oxazolidinonessuch as linezolid, posizolid, radezolid, and torezolid; teixobactin,clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide,isoniazid, pyrazinamide, rifabutin, arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin (or dalfopristin), thiamphenicol, tigecycline, tinidazole,trimethoprim, alatrofloxacin, fidaxomicin, nalidixic acid, rifampin,derivatives and combination thereof.

In some embodiments, the Bacteroides is resistant to an antibiotic, suchas β-lactams, aminoglycosides, erythromycin or tetracycline. In thesecases, the gene encoding the resistance gene for that antibiotic withinthe Bacteroides can be modified to make the Bacteroides susceptible tothe antibiotic. In a further embodiment, the modified Bacteroides isthen treated with the specific antibiotic.

Measurement of Bacterial Elimination

The elimination of bacteria can be assessed by comparison with andwithout (control sample) the antibacterial treatment either in vitro orin vivo. Untreated samples can serve as control samples. The comparisonis preferably performed by assessing the percentage of bacteria beforeand after the antibacterial treatment at at least two timepoints anddetermining a reduced amount of the targeted bacteria at latertimepoint, for example, as described in the examples.

Comparison in vitro can be performed by growing the bacteria in solid orliquid culture and determining the percentages or levels of a bacteriaover time. The percentages or levels can be determined by routinediagnostic procedures including ELISA, PCR, High Resolution Melting, andnucleic acid sequencing.

Comparison in vivo can be performed by collecting samples (e.g., stoolor swab) over time and determining the percentages or levels of abacteria over time. The percentages can be determined by routinediagnostic procedures employing immunodetection (e.g. ELISA), nucleicacid amplification (e.g., PCR), High Resolution Melting, and nucleicacid sequencing.

Preferred levels of elimination of bacteria are at least 70%, 80%, 90%,95%, 97%, 98%, 99%, 99.9%, 99.99%, and 100% of the starting levels of abacteria.

Enzymes/Systems for Inducing Modifications

In some embodiments, the genetic modification is made with one or moreof the following enzymes/systems:

A) Cytosine base editors (CBE) and Adenosine base editors (ABE), forexample as described in Rees et al., Nat Rev Genet. 2018 Dec.; 19(12):770-788, which is hereby incorporated by reference.

So far there are seven types of DNA base editors described:

-   -   Cytosine Base Editor (CBE) that convert C:G into T:A (Komor, A        et al. Nature 533:420-4. (2016))    -   Adenine Base Editor (ABE) that convert A:T into G:C        (Gaudelli, N. M. et al. Nature 551(7681) 464-471 (2017))    -   Cytosine Guanine Base Editor (CGBE) that convert C:G into G:C        (Chen, L et al. Precise and programmable C:G to G:C base editing        in genomic DNA. Biorxiv (2020); Kurt, I et al. Nature        Biotechnology 39: 41-46(2021))    -   Cytosine Adenine Base Editor (CABE) that convert C:G into A:T        (Zhao, D et al. Nature Biotechnology 39:35-40(2021))    -   Adenine Cytosine Base Editor (ACBE) that convert A:T into C:G        (WO2020181180)    -   Adenine Thymine Base Editor (ATBE) that convert A:T into T:A        (WO2020181202)    -   Thymine Adenine Base Editor (TABE) that convert T:A into A:T        (WO2020181193; WO2020181178; WO2020181195)

Base editors differ in the base modification enzymes. CBE rely on ssDNAcytidine deaminase among which: APOBEC1, rAPOBEC1, APOBEC1 mutant orevolved version (evoAPOBEC1), and APOBEC homologs (APOBEC3A (eA3A),Anc689), Cytidine deaminase 1 (CDA1), evoCDA1, FERNY, evoFERNY.

ABE rely on deoxyadenosine deaminase activity of a tandem fusionTadA-TadA* where TadA* is an evolved version of TadA, an E. coli tRNAadenosine deaminase enzyme, able to convert adenosine into Inosine onssDNA. TadA* include TadA-8a-e and TadA-7.10.

Except from base modification enzyme there has been also modificationsimplemented to base editor to increase editing efficacy, precision andmodularity:

-   -   the addition of one or two uracil DNA glycosylase inhibitor        domain (UGI) to prevent base excision repair mechanism to revert        base edition    -   the addition of Mu-GAM that decrease insertion-deletion rate by        inhibiting Non-homologous end joining mechanism in the cell        (NHEJ)    -   the use of nickase active Cas9 (nCas9 D10A) that, by creating        nicks on the non-edited strand favors its repair and        consequently the fixation of the edited base.    -   the use of diverse Cas proteins from for example different        organisms, mutants with different PAM motifs or different        fidelity or different family (e.g. Cas12a).

Non-limiting examples of DNA-based editor proteins include BE1, BE2,BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE,EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM,Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Cas12a-BE, Ea3A-BE3,A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE,VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRY-CBE, SpG-CBE4, SpG-ABE,SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4, enAsBE1.1, enAsBE1.2,enAsBE1.3, enAsBE1.4, AsBE1.1, AsBE1.4, CRISPR-Abest, CRISPR-Cbest,eA3A-BE3, AncBE4.

Cytosine Guanine Base Editors (CGBE) consist of a nickase CRISPR fusedto:

-   -   a. A cytosine deaminase (rAPOBEC) and base excision repair        proteins (e.g. rXRCC1). (Chen, L et al. Precise and programmable        C:G to G:C base editing in genomic DNA. Biorxiv (2020); Chen et        al. Programmable C:G to G:C genome editing with        CRISPR-Cas9-directed base excision repair proteins. Nature        Communications 12:1384 (2021)).    -   b. A rat APOBEC1 variant (R33A) protein and an E. coli-derived        uracil DNA N-glycosylase (eUNG) (Kurt, I et al. Nature        Biotechnology 39: 41-46(2021)).

Cytosine Adenine Base Editors (CABE) consist of a Cas9 nickase, acytidine deaminase (e.g. AID), and a uracil-DNA glycosylase (Ung) (Zhao,D et al. Nature Biotechnology 39:35-40(2021)).

ACBE include a nucleic acid programmable DNA-binding protein and anadenine oxidase (WO2020181180).

ATBE consist of a Cas9 nickase and one or more adenosine deaminase or anoxidase domain (WO2020181202).

TABE consist of a Cas9 nickase and an adenosine methyltransferase, athymine alkyltransferase, or an adenosine deaminase domain(WO2020181193; WO2020181178; WO2020181195).

Base editor molecules can also consist of two or more of the abovelisted editor enzymes fused to a Cas protein (e.g. combination of an ABEand CBE). These biomolecules are named dual base editors and enable theediting of two different bases (Grunewald, J et al. Nature Biotechnology38:861-864(2020); Li, C et al. Nature Biotechnology 38:875-882(2020)).

B) Prime editors (PE), as described in Anzalone et al. Nature576:149-157 (2019) which is hereby incorporated by reference, consist ofa nCas9 fused to a reverse transcriptase used in combination with aprime editing RNA (pegRNA; a guide RNA that includes a template regionfor reverse transcription).

Prime Editing allows introduction of insertions, deletions (indels), and12 base-to-base conversions. Prime editing relies on the ability of areverse transcriptase (RT), fused to a Cas nickase variant, to convertRNA sequence brought by a prime editing guide RNA (pegRNA) into DNA atthe nick site generated by the Cas protein. The DNA flap generated fromthis process is then included or not in the targeted DNA sequence.

Prime Editing Systems Include:

-   -   a Cas nickase variant such as Cas9-H840A fused to a reverse        transcriptase domain such as M-MLV RT or its mutant version        (M-MLV RT(D200N), M-MLV RT(D200N/L603W), M-MLV        RT(D200N/L603W/T330P/T306K/W313F)    -   a prime editing guide RNA (pegRNA)

To favor editing, the prime editing system can include the expression ofan additional sgRNA targeting the Cas nickase activity towards thenon-edited DNA strand ideally only after the resolution of the editedstrand flap by designing the sgRNA to anneal with the edited strand butnot with the original strand.

Non-limiting examples of prime editing systems include PE1, PE1-M1,PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b.

C) Cas9 Retron preciSe Parallel Editing via homologY (‘CRISPEY’), aretron RNA fused to the sgRNA and expressed together with Cas9 and theretron proteins including at least the reverse transcriptase (Sharon, E.et al. Cell 175, 544-557.e16 (2018)).

D) The SCRIBE strategy: a retron system expressed in combination with arecombinase promoting the recombination of single stranded DNA, alsoknown as single stranded annealing proteins (SSAPs) (Farzadfard, F. &Lu, T. K. Science 346, 1256272 (2014)). Such recombinases include butare not limited to phage recombinases such as lambda red, recET, Sak,Sak4, and newly described SSAPs described in Wannier, T. M. et al.(Improved bacterial recombineering by parallelized protein discovery.Biorxiv 2020.01.14.906594 (2020) doi:10.1101/2020.01.14.906594), whichis hereby incorporated by reference.

E) The targetron system based on group II introns described in Karberg,M. et al. Nat Biotechnol 19, 1162-7 (2001), which is hereby incorporatedby reference, and which has been adapted to many bacterial species.

F) Other retron based gene targeting approaches are described in Simonet al. Nucleic Acids Res 47, 11007-11019 (2019) which is herebyincorporated by reference.

G) CRISPR-Cas. The CRISPR system contains two distinct elements, i.e. i)an endonuclease, in this case the CRISPR associated nuclease (Cas or“CRISPR associated protein”) and ii) a guide RNA. Depending on the typeof CRISPR system, the guide RNA may be in the form of a chimeric RNAwhich consists of the combination of a CRISPR (crRNA) bacterial RNA anda tracrRNA (trans-activating RNA CRISPR)¹⁶. The guide RNA combines thetargeting specificity of the crRNA corresponding to the “spacingsequences” that serve as guides to the Cas proteins, and theconformational properties of the tracrRNA in a single transcript. Whenthe guide RNA and the Cas protein are expressed simultaneously in thecell, the target genomic sequence can be permanently interrupted (andcausing disappearance of the targeted and surrounding sequences and/orcell death, depending on the location) or modified. The modification maybe guided by a repair matrix.

The CRISPR system includes two main classes depending on the nucleasemechanism of action:

-   -   Class 1 is made of multi-subunit effector complexes and includes        type I, Ill and IV    -   Class 2 is made of single-unit effector modules, like Cas9        nuclease, and includes type II (II-A, II-B, II-C, II-C variant),        V (V-A, V-B, V-C, V-D, V-E, V-U1, V-U2, V-U3, V-U4, V-U5) and VI        (VI-A, VI-B1, VI-B2, VI-C, VI-D)

The sequence of interest according to the present invention comprises anucleic acid sequence encoding Cas protein. A variety of CRISPR enzymesare available for use as a sequence of interest on the plasmid accordingto the present invention. In some embodiments, the CRISPR enzyme is aType II CRISPR enzyme, a Type II-A or Type II-B CRISPR enzyme. Inanother embodiment, the CRISPR enzyme is a Type I CRISPR enzyme or aType III CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzesDNA modification. In some other embodiments, the CRISPR enzyme catalyzesRNA modification. For instance, Cas13-deaminase fusions have been usedfor RNA base editing thus modifying RNA (David B T Cox et al, Science,358 (6366) p. 1019-1027, 2017 Nov. 24). In one embodiment, the CRISPRenzymes may be coupled to a guide RNA or single guide RNA (sgRNA). Incertain embodiments, the guide RNA or sgRNA targets a gene selected fromthe group consisting of an antibiotic resistance gene, virulence proteinor factor gene, toxin protein or factor gene, a bacterial receptor gene,a membrane protein gene, a structural protein gene, a secreted proteingene, a gene expressing resistance to a drug in general and a genecausing a deleterious effect to the host. Preferably, the CRISPR enzymemakes a double strand break. In some embodiments, the CRISPR enzymemakes a single strand break or nicks. In some embodiments, the CRISPRenzyme does not make any break in the DNA or RNA. In one embodiment, aCas13-deaminase fusion is used to base edit an RNA.

The sequence of interest may comprise a nucleic acid sequence encoding aguide RNA or sgRNA to guide the Cas protein endogenous to the targetedbacteria, alone or in combination with a Cas protein and/or a guide RNAencoded by the payload.

Non-limiting examples of Cas proteins as part of a multi-subuniteffector or as a single-unit effector include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Cas11 (SS), Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d(CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2),Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Csc1, Csc2, Cse1, Cse2,Csy1, Csy2, Csy3, Csf1, Csf2, Csf3, Csf4, Csm1, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx13, Csx1, Csx15, SdCpf1, CmtCpf1, TsCpf1,CmaCpf1, PcCpf1, ErCpf1, FbCpf1, UbcCpf1, AsCpf1, LbCpf1, homologuesthereof, orthologues thereof, variants thereof, or modified versionsthereof. In some embodiments, the CRISPR enzyme cleaves both strands ofthe target nucleic acid at the Protospacer Adjacent Motif (PAM) site.

In various embodiments, the invention encompasses fusion proteinscomprising a Cas9 (e.g., a Cas9 nickase) domain and a deaminase domain.In some embodiments, the fusion protein comprises Cas9 and a cytosinedeaminase enzyme, such as APOBEC enzymes, or adenosine deaminaseenzymes, such as ADAT enzymes, for example as disclosed in U.S. PatentPubl. 2015/0166980, which is hereby incorporated by reference. In oneembodiment, the deaminase is an ACF1/ASE deaminase.

In various embodiments, the APOBEC deaminase is selected from the groupconsisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase,APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3Fdeaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In variousembodiments, the fusion protein comprises a Cas9 domain, a cytosinedeaminase domain, and a uracil glycosylase inhibitor (UGI) domain.

In one embodiment, the deaminase is an adenosine deaminase thatdeaminate adenosine in DNA, for example as disclosed in U.S. Pat. No.10,113,163, which is hereby incorporated by reference. In someembodiments, the fusion proteins further comprise a nuclear localizationsequence (NLS), and/or an inhibitor of base repair, such as, a nucleasedead inosine specific nuclease (dISN), for example as disclosed in U.S.Pat. No. 10,113,163. In various embodiments, the invention encompassesfusion proteins comprising a catalytically impaired Cas9 endonucleasefused to an engineered reverse transcriptase, programmed with a primeediting guide RNA (pegRNA) that both specifies the target site andencodes the desired edit, for example as described in Anzalone et al.,Nature, Vol. 576, pages 149-157 (2019), which is hereby incorporated byreference.

In a particular embodiment, the CRISPR enzyme is any Cas9 protein, forinstance any naturally-occurring bacterial Cas9 as well as any variants,homologs or orthologs thereof.

By “Cas9” is meant a protein Cas9 (also called Csn1 or Csx12) or afunctional protein, peptide or polypeptide fragment thereof, i.e.capable of interacting with the guide RNA(s) and of exerting theenzymatic activity (nuclease) which allows it to perform thedouble-strand cleavage of the DNA of the target genome. “Cas9” can thusdenote a modified protein, for example truncated to remove domains ofthe protein that are not essential for the predefined functions of theprotein, in particular the domains that are not necessary forinteraction with the gRNA(s). In some embodiments, the CAS9 is a dCas9(dead-Cas9) or nCas9 (nickase Cas9) lacking double stranded DNA cleavageactivity.

The sequence encoding Cas9 (the entire protein or a fragment thereof) asused in the context of the invention can be obtained from any known Cas9protein (Fonfara et al., Nucleic Acids Research 42, 2577-2590 (2014);Koonin et al., Current Opinion in Microbiology 37, 67-78 (2017)).Examples of Cas9 proteins useful in the present invention include, butare not limited to, Cas9 proteins of Streptococcus pyogenes (SpCas9),Streptococcus thermophiles (St1Cas9, St3Cas9), Streptococcus mutans,Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9),Francisella novicida (FnCas9) and Neisseria meningitides (NmCas9).

The sequence encoding Cpf1 (Cas12a) (the entire protein or a fragmentthereof) as used in the context of the invention can be obtained fromany known Cpf1 (Cas12a) protein (Koonin et al., Current Opinion inMicrobiology 37, 67-78 (2017)). Examples of Cpf1 (Cas12a) proteinsuseful in the present invention include, but are not limited to, Cpf1(Cas12a) proteins of Acidaminococcus sp, Lachnospiraceae bacteriu andFrancisella novicida.

The sequence encoding Cas13a (the entire protein or a fragment thereof)as used in the context of the invention can be obtained from any knownCas13a (C2c2) protein (Abudayyeh et al., Nature 550, 280 (2017)).Examples of Cas13a (C2c2) proteins useful in the present inventioninclude, but are not limited to, Cas13a (C2c2) proteins of Leptotrichiawadei (LwaCas13a).

The sequence encoding Cas13d (the entire protein or a fragment thereof)as used in the context of the invention can be obtained from any knownCas13d protein (Yan et al. (2018) Mol Cell 70(2):327-339). Examples ofCas13d proteins useful in the present invention include, but are notlimited to, Cas13d proteins of Eubacterium siraeum and Ruminococcus sp.

In some embodiments, other programmable nucleases can be used. Theseinclude an engineered TALEN (Transcription Activator-Like EffectorNuclease) and variants, engineered zinc finger nuclease (ZFN) variants,natural, evolved or engineered meganuclease or recombinase variants, andany combination or hybrids of programmable nucleases. Thus, theprogrammable nucleases provided herein may be used to selectively modifya Bacteroides sp. DNA encoding a gene of interest.

In some embodiments, the genetic modification is made at the RNA level.RNA base editing is based on the same principle as DNA base editing: anenzyme catalyzing the conversion of a RNA base into another must bebrought close to the target base to perform its conversion locally. Inone embodiment, the enzyme used for RNA editing is an adenosinedeaminase from ADAR family that converts Adenosine into Inosine in dsRNAstructure. Several seminal studies used this specificity for dsRNA andfused the ADAR deaminase domain (ADAR_(DD)) to an antisense oligo inorder to program local RNA base editing. More recently the ability ofsome CRISPR-Cas systems to bind RNA molecules was repurposed into RNAediting. Using catalytically dead Cas13b enzyme (dPspCas13b) fused to ahyperactive mutant of ADAR2 deaminase domain (ADAR2_(DD)-E488Q forREPAIRv1 and ADAR2_(DD)-E488Q-T375G for REPAIRv2) Cox et al improvedspecificity and efficiency compare to previous RNA editing strategies.

Non-limiting examples of RNA based editor proteins include REPAIRv1,REPAIRv2.

Vectors for Inducing Modifications

In various embodiments, one or more of the following vectors can be usedto eliminate or reduce deleterious Bacteroides sp., in particular tointroduce the exogenous enzyme that results in a genetic modification orto introduce an endolysin:

-   -   engineered phages,    -   engineered bacteria,    -   plasmid (eg, a conjugative plasmid capable of transfer into a        host cell), phage, phagemid or prophage.    -   Each vector may be as described above, eg, a phage capable of        infecting a host cell or conjugative plasmid capable of        introduction into a host cell, which can be introduced either by        a phage particle (engineered or wild-type phage) via injection        or by a donor bacteria via conjugation. In an example, the        vectors are in combination with an antibiotic agent (eg, a        beta-lactam antibiotic) and/or with any other agent.    -   A bacteriophage for modifying a naturally occurring bacteria in        situ comprising a nucleic acid encoding a gene editing        enzyme/system for transformation of a target bacteria in a mixed        bacterial population wherein said gene editing enzyme/system        modifies the genome of said target bacteria, but does not lead        to the death of the target bacteria.

The invention encompasses the use of these vectors wherein the geneediting enzyme/system targets a gene within the target bacteria encodinga protein which is directly or indirectly responsible for a disease ordisorder, in particular myocarditis or DCM. In a particular embodiment,the gene editing enzyme/system targets a β-galactosidase gene.

Bacterial viruses (also called bacteriophages or phages) are smallviruses displaying the ability to infect and kill bacteria while they donot affect cells from other organisms. Initially described almost acentury ago by William Twort, and independently discovered shortlythereafter by Felix d′Herelle, more than 6000 different bacterialviruses have been discovered so far and described morphologically. Thevast majority of these viruses are tailed while a small proportion, arepolyhedral, filamentous or pleomorphic. They may be classified accordingto their morphology, their genetic content (DNA vs. RNA), their specifichost, the place where they live (marine virus vs. other habitats), andtheir life cycle. As intracellular parasites of bacterial cells, phagesdisplay different life cycles within the bacterial host: lytic,lysogenic, pseudo-lysogenic, and chronic infection. Lytic phages, oncetheir DNA injected into their host, replicate their own genome andproduce new viral particles at the expense of the host. Indeed, theycause lysis of the host bacterial cell as a normal part of the finalstage of their life cycles to liberate viral particles. Temperate phagescan either replicate by means of the lytic life cycle and cause lysis ofthe host bacterium, or they can incorporate their DNA into the hostbacterial DNA and become non-infectious prophages (lysogenic cycle). Insome embodiments, lytic phages are used.

Unlike classical chemically-based antibiotics that are active against abroad spectrum of bacterial species, a bacteriophage can infect and killonly a small number of different closely-related bacteria.

The use of packaged phagemids) allows to have a defined and controlledway of killing the host. Example of packaged phagemids encodingCRISPR-Cas9 or toxins have shown promising results in killing targetedbacterial population (Bikard et al., 2012, Cell Host & Microbe 12,177-186; Jiang et al., 2013, Nat Biotechnol 31, 233-239; Krom et al.,2015, Nano Letters 15, 4808-4813; Bikard et al, 2014, Nat Biotech 11,Vol. 32, Citorik, R et al, 2014, Nat Biotech 11, Vol. 32).

Sequence of Interest Under the Control of the Promoter

The vector can comprise a sequence of interest under the control of apromoter.

In one embodiment, the sequence of interest is a programmable nucleasecircuit to be delivered to the targeted bacteria. This programmablenuclease circuit may be able to mediate in vivo sequence-specificelimination of bacteria that contain a target gene of interest. Someembodiments of the present disclosure relate to engineered variants ofthe Type II CRISPR-Cas (Clustered Regularly Interspaced ShortPalindromic Repeats-CRISPR-associated) system of Streptococcus pyogenes.Other programmable nucleases that can be used include other CRISPR-Cassystems, engineered TALEN (Transcription Activator-Like EffectorNuclease) variants, engineered zinc finger nuclease (ZFN) variants,natural, evolved or engineered meganuclease or recombinase variants, andany combination or hybrids of programmable nucleases.

Other sequences of interest, preferably programmable, can be added tothe payload so as to be delivered to targeted bacteria.

Preferably, the sequence of interest circuit added to the payload leadsto bacteria death. The sequence of interest may comprise proteins andenzymes achieving a useful function such as modifying the metabolism ofthe bacteria, the composition of its environment or affecting the host.

In a particular embodiment, the nucleic acid sequence of interest isselected from the group consisting of a Cas nuclease, a type I nuclease,a type II nuclease, a type V nuclease, a Cas9 nuclease, a Cpf1 nuclease,a Cas3 nuclease, a Cms1 nuclease, a MAD nuclease, a guide RNA, a singleguide RNA (sgRNA), a CRISPR locus, a gene expressing an enzyme such as anuclease or a kinase, a TALEN, a ZFN, a meganuclease, a recombinase, abacterial receptor, a membrane protein, a structural protein, a secretedprotein, a gene expressing resistance to an antibiotic or to a drug ingeneral, a gene expressing a toxic protein or a toxic factor and a geneexpressing a virulence protein or a virulence factor, a bacterialsecretory protein or transporter, a bacterial pore or any of theircombination. These proteins can also be modified or engineered toinclude extra features, like the addition or removal of a function (e.g.dCas9), the addition of a secretion signal to a protein not normallysecreted, the addition of an exogenous peptide in a loop as non-limitingexamples.

Targeted Bacteria

The Bacteroides targeted by a composition of the invention can bepresent in vivo, in a mammalian organism, or in vitro, for example inliquid or solid culture.

A microbiome may comprise a variety of endogenous Bacteroides species,any of which may be targeted in accordance with the present disclosure.In some embodiments, the species of targeted Bacteroides bacterial cellsmay depend on the type of bacteriophages being used for preparing thebacterial virus particles. For example, some bacteriophages exhibittropism for, or preferentially target, specific host species ofbacteria. Other bacteriophages do not exhibit such tropism and may beused to target a number of different genus and/or species of endogenousbacterial cells.

Examples of Bacteroides species include, without limitation, B.acidifaciens, B. barnesiaes, B. caccae, B. caecicola, B.caecigallinarum, B. cellulosilyticus, B. cellulosolvens, B. clarus, B.coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. distasonis, B.eggerthii, B. gracilis, B. faecichinchillae, B. faecis, B. finegoldii,B. fluxus, B. fragilis, B. galacturonicus, B. gallinaceumi, B.gallinarum, B. goldsteinii, B. graminisolvens, B. helcogene, B.intestinalis, B. luti, B. massiliensis, B. melaninogenicus, B. nordii,B. oleiciplenus, B. oris, B. ovatus, B. paurosaccharolyticus, B.plebeius, B. polypragmatus, B. propionicifaciens, B. putredinis, B.pyogenes, B. reticulotermitis, B. rodentium, B. salanitronis, B.salyersiae, B. sartorii, B. sedimenti, B. stercoris, B. suis, B. tectus,B. thetaiotaomicron, B. uniformis, B. vulgatus, B. xylanisolvens, and B.xylanolyticus.

Thus, bacterial virus particles may target (e.g., specifically target) abacterial cell from any one or more of the foregoing species of bacteriato specifically deliver the plasmid according to the invention.

In preferred embodiments, the targeted bacterial cells are, withoutlimitation, Bacteroides faecis, Bacteroides thetaiotaomicron,Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, andBacteroides fragilis.

In particular embodiments, the targeted bacterial cells are, withoutlimitation, Bacteroides sp. bacterial which produce MYH6 mimic peptides(in particular mimics of MYH6₆₁₄₋₆₂₉ or MYH6₆₁₄₋₆₂₈ peptides, typicallyof sequence SEQ ID NO: 10, 16 or 17), more particularly which produceβ-galactosidase (typically of sequence SEQ ID NO: 1), more particularlywhich produce β-gal₁₁₋₂₅ peptide (typically of sequence SEQ ID NO: 11,12, 18, 19, 20, 21 or 22).

Bacterial Viruses

The bacterial virus particles are prepared from bacterial virus. Thebacterial viruses can be chosen in order to be able to introduce theplasmid into the targeted bacteria.

Bacterial viruses are preferably bacteriophages. Bacteriophages areobligate intracellular parasites that multiply inside bacteria byco-opting some or all of the host biosynthetic machinery. Phage genomescome in a variety of sizes and shapes (e.g., linear or circular). Mostphages range in size from 24-200 nm in diameter. Phages contain nucleicacid (i.e., genome) and proteins, and may be enveloped by a lipidmembrane. Depending upon the phage, the nucleic acid genome can beeither DNA or RNA, and can exist in either circular or linear forms. Thesize of the phage genome varies depending upon the phage. The simplestphages have genomes that are only a few thousand nucleotides in size,while the more complex phages may contain more than 100,000 nucleotidesin their genome, and in rare instances more than 1,000,000. The numberand amount of individual types of protein in phage particles will varydepending upon the phage.

A non-exhaustive listing of known Bacteroides-specific bacteria virusesis presented in the following paragraphs. Synonyms and spelling variantsare indicated in parentheses. Homonyms are repeated as often as theyoccur (e.g., D, D, d). Unnamed phages are indicated by “NN” beside theirgenus and their numbers are given in parentheses.

Bacteria of the genus Bacteroides can be infected by the followingphages: crAss-phage, ad l2, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8,FI, βI, φAl, φBrOI, φBrO2, 11, 67.1, 67.3, 68.1, mt-Bacteroides (3),Bf42, Bf71, HN-Bdellovibrio (1) and BF-41.

In some embodiment the vectors disclosed herein may be used incombination with prebiotics. Prebiotics include, but are not limited to,amino acids, biotin, fructo-oligosaccharide, galacto-oligosaccharides,hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan),inulin, chitin, lactulose, mannan oligosaccharides,oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic andcarrageenan), oligofructose, oligodextrose, tagatose, resistantmaltodextrins (e.g., resistant starch), trans-galactooligosaccharide,pectins (e.g., xylogalacturonan, citrus pectin, apple pectin, andrhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber,pea fiber, corn bran, and oat fiber) and xylooligosaccharides.

In other embodiment, the vectors, antibiotics, bacteriocins and/orendolysins disclosed herein may be used in combination with bacteriaand/or engineered bacteria and/or probiotics. Probiotics include, butare not limited to lactobacilli, bifidobacteria, streptococci,enterococci, propionibacteria, saccharomycetes, lactobacilli,bifidobacteria, or proteobacteria.

In one embodiment, said bacteria and/or engineered bacteria, inparticular engineered Bacteroides sp. and/or probiotics do not produceMYH6 mimic peptides (in particular mimics of MYH6₆₁₄₋₆₂₉ or MYH6₆₁₄₋₆₂₈peptides, typically of sequence SEQ ID NO: 10, 16 or 17), moreparticularly do not produce β-gal₁₁₋₂₅ peptide (typically of sequenceSEQ ID NO: 11, 12, 18, 19, 20, 21 or 22), and preferably present acompetitive advantage over Bacteroides sp. to be reduced or eliminated.

In a particular embodiment, said probiotic is an engineered probiotic.

In a particular embodiment, said engineered bacteria and/or probioticsfurther comprise an heterologous gene or gene circuit required to importand/or metabolize a particular exogenous food source, such as anexogenous sugar.

In a particular embodiment, said engineered bacteria and/or probioticsmay further be engineered to be insensitive to the vectors, phages,antibiotics, bacteriocins and/or endolysins used herein.

In a particular embodiment, the vector, phage, antibiotic, bacteriocinand/or endolysin disclosed herein is used in combination with (i) anengineered bacteria and/or probiotic further comprising an heterologousgene or gene circuit required to import and/or metabolize a particularexogenous food source and (ii) said exogenous food source.

The combined use of the vector disclosed herein and the herein disclosedengineered bacteria and/or probiotic and the corresponding exogenousfood source typically enables said engineered bacteria and/or probioticto fill the niche left empty by the bacteria, in particular theBacteroides bacteria killed by the use of the vector of the invention.

Said bacteria and/or engineered bacteria and/or probiotic can typicallybe administered before, simultaneously with or after said vector, phage,antibiotic, bacteriocin and/or endolysin.

Screening Methods

The invention encompasses methods for screening for geneticmodifications in Bacteroides sp. In one embodiment, the method comprisesadministering a vector designed to genetically modify at least one baseof a DNA of interest in a gene of a Bacteroides sp., to a subject,subsequently collecting a bacterial sample from the subject,quantitating the level of bacteria containing a genetic modification insaid at least one base of a DNA of interest in said bacterial sample.The genetic modification can be the insertion of an exogenous DNA, forexample, encoding an endolysin. The method can further comprisequantitating the level of bacteria not containing a genetic modificationin said at least one base of a DNA of interest.

In one embodiment, the proportion of Bacteroides sp. modified vsnon-modified bacteria is quantified. Preferred percentages of bacteriawith the genetic modification are at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 97%, 98%, 99%, 99.9%, 99.99%, and 100%.

In one embodiment, the number of non-modified endogenous bacteria isquantified prior to administering a vector. The patient can also bepre-screened to determine the genetic signature of the strains thepatient carries. This will allow selection of an appropriate capsid todeliver the therapeutic payload based on the genetic signature of thestrains the patient carries.

In preferred embodiments, the vector is in a pharmaceutical orveterinary composition. Preferably the vector is a bacteriophage.

The vector can be administered to the subject by any administrationtechnique known in the art, depending on the vector and the targetbacteria's expected location in or on the subject.

The bacterial sample can be collected by any means known in the art,such as biopsy, blood draw, urine sample, stool sample, or oral/nasalswab, etc.

The level of bacteria containing or not containing a geneticmodification in a base of a DNA of interest can be determined by anytechnique known to the skilled artisan, such as routine diagnosticprocedures including ELISA, PCR, High Resolution Melting, and nucleicacid sequencing.

The invention encompasses methods for determining the efficiency of avector at inducing genetic mutations in situ. In one embodiment, themethod comprises administering a vector designed to genetically modifyat least one base of a DNA of interest in a gene of a naturallyoccurring bacteria, to a subject, subsequently collecting a bacterialsample from the subject, quantitating the level of bacteria containing agenetic modification in said at least one base of a DNA of interest andquantitating the level of bacteria not containing a genetic modificationin said at least one base of a DNA of interest in said bacterial sample.

In preferred embodiments, the vector is in a pharmaceutical orveterinary composition. Preferably the vector is a bacteriophage.

The vector can be administered to the subject by any administrationtechnique known in the art, depending on the vector and the targetbacteria's expected location in or on the subject.

The bacterial sample can be collected by any means known in the art,such as biopsy, blood draw, urine sample, stool sample, or oral/nasalswab, etc.

The level of bacteria containing or not containing a geneticmodification in a base of a DNA of interest can be determined by anytechnique known to the skilled artisan, such as routine diagnosticprocedures including ELISA, PCR, High Resolution Melting, and nucleicacid sequencing.

The invention encompasses methods for determining the effect of agenetic mutation on bacterial growth. In one embodiment, the methodcomprises administering a vector designed to genetically modify at leastone base of a DNA of interest in a gene of a Bacteroides sp., to asubject, subsequently collecting at least two sequential bacterialsamples from the subject, quantitating the level of bacteria containinga genetic modification in said at least one base of a DNA of interestand quantitating the level of bacteria not containing a geneticmodification in said at least one base of a DNA of interest in saidbacterial samples.

In preferred embodiments, the vector is in a pharmaceutical orveterinary composition. Preferably the vector is a bacteriophage.

The vector can be administered to the subject by any administrationtechnique known in the art, depending on the vector and the targetbacteria's expected location in or on the subject.

The bacterial samples can be collected by any means known in the art,such as biopsy, blood draw, urine sample, stool sample, or oral/nasalswab, etc. The samples can be collected at any sequential time points.Preferably, the time between these collections is at least 3, 6, 12, 24,48, 72, 96 hours or 7, 14, 30, 60, 120, or 365 days.

The level of bacteria containing or not containing a geneticmodification in a base of a DNA of interest can be determined by anytechnique known to the skilled artisan, such as routine diagnosticprocedures including ELISA, PCR, High Resolution Melting, and nucleicacid sequencing.

All of the screening methods of the invention can use any of the vectorsand enzymes/systems of the invention to screen for any of the geneticmodification of the invention.

All of the screening methods of the invention can further include a stepof comparing the level of bacteria containing a genetic modification ina base of a DNA of interest with the level of bacteria not containing agenetic modification the base of a DNA of interest in a bacterialsample.

All of the screening methods of the invention can further include a stepof contacting the vector with bacteria in liquid or solid culture andquantitating the level of bacteria containing a genetic modification insaid at least one base of a DNA of interest in said bacterial sample.The method can further comprise quantitating the level of bacteria notcontaining a genetic modification in said at least one base of a DNA ofinterest.

In one embodiment, the method comprises providing a vector designed togenetically modify at least one base of a DNA of interest in a gene of aBacteroides sp. The method can further comprise contacting the vectorwith a Bacteroides sp. in liquid or solid culture and quantitating thelevel of Bacteroides sp. containing a genetic modification in said atleast one base of a DNA of interest in said bacterial sample. The methodcan further comprise quantitating the level of bacteria not containing agenetic modification in said at least one base of a DNA of interest. Thelevels of bacteria containing a genetic modification in a base of a DNAof interest can be compared with the level of bacteria not containing agenetic modification the base of a DNA of interest over time in theculture. Preferably, the time between these comparisons is at least 1,2, 3, 4, 5, 6, 12, 24, 48, 72, or 96 hours.

The invention encompasses methods for determining the efficiency of abacteria, engineered bacteria, antibiotic, bacteriocin or endolysin atreducing or killing Bacteroides sp. in situ. In one embodiment, themethod comprises providing a bacteria, engineered bacteria, antibiotic,bacteriocin or endolysin, contacting the bacteria, engineered bacteria,antibiotic, bacteriocin or endolysin with a Bacteroides sp. in situ, andquantitating the level of Bacteroides sp. killed or reduced by thebacteria, engineered bacteria, antibiotic, bacteriocin or endolysin. Insome embodiments, the endolysin or bacteriocin is encoded by a vector,such as a bacteriophage. The method can further comprise quantitatingthe level of Bacteroides not killed or reduced by the bacteria,engineered bacteria, antibiotic, bacteriocin or endolysin. The levels ofbacteria can be compared over time. Preferably, the time between thesecomparisons is at least 1, 2, 3, 4, 5, 6, 12, 24, 48, 72, or 96 hours.

Pharmaceutical and Veterinary Compositions and In Situ AdministrationMethods

The invention encompasses pharmaceutical and veterinary compositionscomprising the vectors, bacteria, engineered bacteria, antibiotics,bacteriocins and/or endolysins of the invention.

The invention encompasses a pharmaceutical agent which reduces theamount of Bacteroides sp. in a subject.

The invention encompasses in situ administration of the pharmaceuticalor veterinary composition to the bacteria in a subject. Any method knownto the skilled artisan can be used to contact the composition with thebacterial target in situ.

In one embodiment, the composition comprises an effective amount of anantibiotic, phage, recombinant phage, packaged phagemid, bacteria,engineered bacteria, bacteriocin or endolysin.

In one embodiment, the antibiotic is selected from streptomycin,vancomycin, clindamycin, metronidazole, sulphadoxine, trimethoprim, orany combination of 1, 2, 3, 4, 5 or 6 of these antibiotics.

In one embodiment, the phage, recombinant phage, packaged phagemidencodes an endolysin or a bacteriocin.

In one embodiment, the phage, recombinant phage, packaged phagemidencodes a nuclease selected from CRISPR-Cas, TALENs and variants, zincfinger nuclease (ZFN) and ZFN variants, natural, evolved or engineeredmeganuclease or recombinase variants.

In one embodiment, the Bacteroides is B. thetaiotaomicron or B. faecis.

The pharmaceutical or veterinary composition according to the inventionmay further comprise a pharmaceutically acceptable vehicle. A solidpharmaceutically acceptable vehicle may include one or more substanceswhich may also act as flavoring agents, lubricants, solubilizers,suspending agents, dyes, fillers, glidants, compression aids, inertbinders, sweeteners, preservatives, dyes, coatings, ortablet-disintegrating agents. Suitable solid vehicles include, forexample calcium phosphate, magnesium stearate, talc, sugars, lactose,dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low meltingwaxes and ion exchange resins.

The pharmaceutical or veterinary composition may be prepared as asterile solid composition that may be suspended at the time ofadministration using sterile water, saline, or other appropriate sterileinjectable medium. The pharmaceutical or veterinary compositions of theinvention may be administered orally in the form of a sterile solutionor suspension containing other solutes or suspending agents (forexample, enough saline or glucose to make the solution isotonic), bilesalts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleateesters of sorbitol and its anhydrides copolymerized with ethylene oxide)and the like. The particles according to the invention can also beadministered orally either in liquid or solid composition form.Compositions suitable for oral administration include solid forms, suchas pills, capsules, granules, tablets, and powders, and liquid forms,such as solutions, syrups, elixirs, and suspensions. Forms useful forenteral administration include sterile solutions, emulsions, andsuspensions.

The bacterial virus particles according to the invention may bedissolved or suspended in a pharmaceutically acceptable liquid vehiclesuch as water, an organic solvent, a mixture of both or pharmaceuticallyacceptable oils or fats. The liquid vehicle can contain other suitablepharmaceutical additives such as solubilizers, emulsifiers, buffers,preservatives, sweeteners, flavouring agents, suspending agents,thickening agents, colours, viscosity regulators, stabilizers orosmo-regulators. Suitable examples of liquid vehicles for oral andenteral administration include water (partially containing additives asabove, e.g. cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols andpolyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.fractionated coconut oil and arachis oil). For parenteraladministration, the vehicle can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid vehicles are useful insterile liquid form compositions for enteral administration. The liquidvehicle for pressurized compositions can be a halogenated hydrocarbon orother pharmaceutically acceptable propellant.

In some embodiments, the invention encompasses pharmaceutical orveterinary composition formulated for delayed or gradual entericrelease. In preferred embodiments, formulations or pharmaceuticalpreparations of the invention are formulated for delivery of the vectorinto the distal small bowel and/or the colon. The formulation can allowthe vector to pass through stomach acid and pancreatic enzymes and bile,and reach undamaged to be viable in the distal small bowel and colon.

In some embodiments, the pharmaceutical or veterinary composition ismicro-encapsulated, formed into tablets and/or placed into capsules,preferably enteric-coated capsules.

In some embodiments, the pharmaceutical or veterinary compositions areformulated for delayed or gradual enteric release, using celluloseacetate (CA) and polyethylene glycol (PEG). In some embodiments, thepharmaceutical or veterinary compositions are formulated for delayed orgradual enteric release using a hydroxypropylmethylcellulose (HPMC), amicrocrystalline cellulose (MCC) and magnesium stearate. thepharmaceutical or veterinary compositions are formulated for delayed orgradual enteric release using e.g., a poly(meth)acrylate, e.g. amethacrylic acid copolymer B, a methyl methacrylate and/or a methacrylicacid ester, or a polyvinylpyrrolidone (PVP).

In some embodiments, the pharmaceutical or veterinary compositions areformulated for delayed or gradual enteric release using arelease-retarding matrix material such as: an acrylic polymer, acellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetableoil, hydrogenated castor oil, polyvinylpyrrolidone, a vinyl acetatecopolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylicacid and methacrylic acid copolymer, a methyl methacrylate copolymer, anethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, anaminoalkyl methacrylate copolymer, a poly(acrylic acid), apoly(methacrylic acid), a methacrylic acid alkylamide copolymer, apoly(methyl methacrylate), a poly(methacrylic acid anhydride), a methylmethacrylate polymer, a polymethacrylate, a poly(methyl methacrylate)copolymer, a polyacrylamide, an aminoalkyl methacrylate copolymer, aglycidyl methacrylate copolymer, a methyl cellulose, an ethylcellulose,a carboxymethylcellulose, a hydroxypropylmethylcellulose, ahydroxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropylcellulose, a crosslinked sodium carboxymethylcellulose, a crosslinkedhydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol,a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenatedfat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax,glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolicacid, a co-polymer of lactic and glycolic acid, carboxymethyl starch,potassium methacrylate/divinylbenzene copolymer, crosslinkedpolyvinylpyrrolidone, polyvinylalcohols, polyvinylalcohol copolymers,polyethylene glycols, non-crosslinked polyvinylpyrrolidone, polyvinylacetates, polyvinylacetate copolymers or any combination thereof.

In some embodiments, the pharmaceutical or veterinary compositions areformulated for delayed or gradual enteric release as described in U.S.Pat. App. Pub. 20110218216, which describes an extended releasepharmaceutical composition for oral administration, and uses ahydrophilic polymer, a hydrophobic material and a hydrophobic polymer ora mixture thereof, with a microenvironment pH modifier. The hydrophobicpolymer can be ethylcellulose, cellulose acetate, cellulose propionate,cellulose butyrate, methacrylic acid-acrylic acid copolymers or amixture thereof. The hydrophilic polymer can be polyvinylpyrrolidone,hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose,polyethylene oxide, acrylic acid copolymers or a mixture thereof. Thehydrophobic material can be a hydrogenated vegetable oil, hydrogenatedcastor oil, carnauba wax, candelilla wax, beeswax, paraffin wax, stearicacid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or and amixture thereof. The microenvironment pH modifier can be an inorganicacid, an amino acid, an organic acid or a mixture thereof.Alternatively, the microenvironment pH modifier can be lauric acid,myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid,oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid,fumaric acid, maleic acid; glycolic acid, lactic acid, malic acid,tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, asalicylic acid, tosylic acid, mesylic acid or malic acid or a mixturethereof.

In some embodiments, the pharmaceutical or veterinary compositions are apowder that can be included into a tablet or a suppository. Inalternative embodiments, a formulation or pharmaceutical preparation ofthe invention can be a ‘powder for reconstitution’ as a liquid to bedrunk or otherwise administered.

In some embodiments, the pharmaceutical or veterinary compositions canbe administered in a cream, gel, lotion, liquid, feed, or aerosol spray.In some embodiments, a bacteriophage is immobilized to a solid surfaceusing any substance known in the art and any technology known in theart, for example, but not limited to immobilization of bacteriophagesonto polymeric beads using technology as outlined in U.S. Pat. No.7,482,115, which is incorporated herein by reference. Phages may beimmobilized onto appropriately sized polymeric beads so that the coatedbeads may be added to aerosols, creams, gels or liquids. The size of thepolymeric beads may be from about 0.1 μm to 500 μm, for example 50 μm to100 μm. The coated polymeric beads may be incorporated into animal feed,including pelleted feed and feed in any other format, incorporated intoany other edible device used to present phage to the animals, added towater offered to animals in a bowl, presented to animals through waterfeeding systems. In some embodiments, the compositions are used fortreatment of surface wounds and other surface infections using creams,gels, aerosol sprays and the like.

In some embodiments, the pharmaceutical or veterinary compositions canbe administered by inhalation, in the form of a suppository or pessary,topically (e.g., in the form of a lotion, solution, cream, ointment ordusting powder), epi- or transdermally (e.g., by use of a skin patch),orally (e.g., as a tablet, which may contain excipients such as starchor lactose), as a capsule, ovule, elixirs, solutions, or suspensions(each optionally containing flavoring, coloring agents and/orexcipients), or they can be injected parenterally (e.g., intravenously,intramuscularly or subcutaneously). For parenteral administration, thecompositions may be used in the form of a sterile aqueous solution whichmay contain other substances, for example enough salts ormonosaccharides to make the solution isotonic with blood. For buccal orsublingual administration the compositions may be administered in theform of tablets or lozenges which can be formulated in a conventionalmanner. In a preferred embodiment, a bacteriophage and/or polypeptide ofthe present invention is administered topically, either as a singleagent, or in combination with other antibiotic treatments, as describedherein or known in the art.

In some embodiments, the pharmaceutical or veterinary compositions canalso be dermally or transdermally administered. For topical applicationto the skin, the pharmaceutical or veterinary composition can becombined with one or a combination of carriers, which can include butare not limited to, an aqueous liquid, an alcohol base liquid, a watersoluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineraloil base, a blend of mineral oil and petrolatum, lanolin, liposomes,proteins carriers such as serum albumin or gelatin, powdered cellulosecarmel, and combinations thereof. A topical mode of delivery may includea smear, a spray, a bandage, a time-release patch, a liquid-absorbedwipe, and combinations thereof. The pharmaceutical or veterinarycomposition can be applied to a patch, wipe, bandage, etc., eitherdirectly or in a carrier(s). The patches, wipes, bandages, etc., may bedamp or dry, wherein the phage and/or polypeptide (e.g., a lysin) is ina lyophilized form on the patch. The carriers of topical compositionsmay comprise semi-solid and gel-like vehicles that include a polymerthickener, water, preservatives, active surfactants, or emulsifiers,antioxidants, sun screens, and a solvent or mixed solvent system. U.S.Pat. No. 5,863,560 discloses a number of different carrier combinationsthat can aid in the exposure of skin to a medicament, and its contentsare incorporated herein.

For intranasal or administration by inhalation, the pharmaceutical orveterinary composition is conveniently delivered in the form of a drypowder inhaler or an aerosol spray presentation from a pressurizedcontainer, pump, spray, or nebuliser with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, carbondioxide, or other suitable gas. In the case of a pressurized aerosol,the dosage unit may be determined by providing a valve to deliver ametered amount. The pressurized container, pump, spray, or nebuliser maycontain a solution or suspension of the active compound, e.g., using amixture of ethanol and the propellant as the solvent, which mayadditionally contain a lubricant, e.g., sorbitan trioleate. Capsules andcartridges (made, for example, from gelatin) for use in an inhaler orinsufflator may be formulated to contain a powder mix of thebacteriophage and/or polypeptide of the invention and a suitable powderbase such as lactose or starch.

For administration in the form of a suppository or pessary, thepharmaceutical or veterinary composition can be applied topically in theform of a gel, hydrogel, lotion, solution, cream, ointment, or dustingpowder. Compositions of the invention may also be administered by theocular route. For ophthalmic use, the compositions of the invention canbe formulated as micronized suspensions in isotonic, pH adjusted,sterile saline, or, preferably, as solutions in isotonic, pH adjusted,sterile saline, optionally in combination with a preservative such as abenzalkonium chloride. Alternatively, they may be formulated in anointment such as petrolatum.

Dosages and desired drug concentrations of the pharmaceutical andveterinary composition compositions of the present invention may varydepending on the particular use. The determination of the appropriatedosage or route of administration is within the skill of an ordinaryphysician. Animal experiments can provide reliable guidance for thedetermination of effective doses in human therapy.

For transdermal administration, the pharmaceutical or veterinarycomposition can be formulated into ointment, cream or gel form andappropriate penetrants or detergents could be used to facilitatepermeation, such as dimethyl sulfoxide, dimethyl acetamide anddimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginalsuppositories can be used. The active compounds can be incorporated intoany of the known suppository bases by methods known in the art. Examplesof such bases include cocoa butter, polyethylene glycols (carbowaxes),polyethylene sorbitan monostearate, and mixtures of these with othercompatible materials to modify the melting point or dissolution rate.

Subject, Regimen and Administration

The subject according to the invention is an animal, preferably amammal, even more preferably a human. However, the term “subject” canalso refer to non-human animals, in particular mammals such as dogs,cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, gerbils,hamsters, chinchillas, rats, mice, guinea pigs and non-human primates,among others, or non-mammals such as poultry, that are in need oftreatment.

The human subject according to the invention may be a human at theprenatal stage, a new-born, a child, an infant, an adolescent or anadult at any age.

In a particular embodiment, the subject is carrying a HLA-DQ haplotypewhich binds to human MYH6₆₁₄₆₂₉ (typically of sequence SEQ ID NO: 17);more particularly (i) a HLA-DQB1*03 variant (in particular encoded byDQB1*03:01, DQB1*03:02, DQB1*03:03, DQB1*03:04, or DQB1*03:05 alleles)and/or (ii) a HLA-DQA1*01:02 or a HLA-DQA1*05:01 variant; stillparticularly a HLA-DQ7 (for example encoded by DQA1*02:01/DQB1*03:01,DQA1*03:01/DQB1*03:01, DQA1*03:03/DQB1*03:01, DQA1*03:01/DQB1*03:04,DQA1*03:02/DQB1*03:04, DQA1*04:01/DQB1*03:01, DQA1*05:05/DQB1*03:01, orDQA1*06:01/DQB1*03:01 alleles), HLA-DQ8 (for example encoded byDQA1*03:01/DQB1*03:02 or DQA1*03:02/DQB1*03:02 alleles) or HLA-DQ9 (forexample encoded by DQA1*02:01/DQB1*03:03 or DQA1*03:02/DQB1*03:03alleles) haplotype.

In a preferred embodiment, the subject has been diagnosed with, or is atrisk of developing myocarditis or DCM. Diagnostic methods of determiningBacteroides sp. infection are well known by the man skilled in the art.

In a particular embodiment, the subject has never received any treatmentprior to the administration of the vectors, delivery vehicles, bacteria,engineered bacteria, antibiotics, bacteriocins or endolysins accordingto the invention, preferably a payload according to the invention,particularly a payload packaged into a delivery vehicle according to theinvention, preferably a packaged plasmid or phagemid into a bacterialvirus particle according to the invention, or of a pharmaceutical orveterinary composition according to the invention.

In a particular embodiment, the subject has already received at leastone line of treatment, preferably several lines of treatment, prior tothe administration of the vectors, delivery vehicles, bacteria,engineered bacteria, antibiotics, bacteriocins or endolysins accordingto the invention, preferably a payload according to the invention,particularly a payload packaged into a delivery vehicle according to theinvention, preferably a packaged plasmid or phagemid into a bacterialvirus particle according to the invention, or of a pharmaceutical orveterinary composition according to the invention.

In a particular embodiment, the subject suffers from cancer. In a moreparticular embodiment, the subject has already received animmune-checkpoint inhibitor treatment, more particularly a combinationof anti-CTLA-4 and anti-PD-1 therapy, in particular to treat cancer.

In a particular embodiment, the subject suffers from an auto-immunedisease, in particular from sarcoidosis, rheumatoid arthritis,dermatomyositis, systemic lupus erythematosus (SLE) or giant cellmyocarditis.

In a more particular embodiment, the subject suffers from systemic lupuserythematosus (SLE). More particularly, the subject carries a mutationin the gene encoding the 3′-5′ DNA exonuclease TREX1.

In a particular embodiment, the subject suffers from a bacterial, viral,parasital, protozoan or fungal infection, which may cause myocarditis.

In a particular embodiment, the subject has already received anantiretroviral treatment, an antipsychotic treatment, an anticancertreatment and/or anti-parasite treatment, such as an anti-malariatreatment.

Preferably, the treatment is administered regularly, preferably betweenevery day and every month, more preferably between every day and everytwo weeks, more preferably between every day and every week, even morepreferably the treatment is administered every day. In a particularembodiment, the treatment is administered several times a day,preferably 2 or 3 times a day, even more preferably 3 times a day.

The duration of treatment with vectors, delivery vehicles, bacteria,engineered bacteria, antibiotics, bacteriocins or endolysins accordingto the invention, preferably a payload according to the invention,particularly a payload packaged into a delivery vehicle according to theinvention, preferably a packaged plasmid or phagemid into a bacterialvirus particle according to the invention, or with a pharmaceutical orveterinary composition according to the invention, is preferablycomprised between 1 day and 20 weeks, more preferably between 1 day and10 weeks, still more preferably between 1 day and 4 weeks, even morepreferably between 1 day and 2 weeks. In a particular embodiment, theduration of the treatment is of about 1 week. Alternatively, thetreatment may last as long as the infection, disorder and/or diseasepersists.

The form of the pharmaceutical or veterinary compositions, the route ofadministration and the dose of administration of vectors, deliveryvehicles, bacteria, engineered bacteria, antibiotics, bacteriocins orendolysins according to the invention, preferably of a payload accordingto the invention, particularly of a payload packaged into a deliveryvehicle according to the invention, preferably of a packaged plasmid orphagemid into a bacterial virus particle according to the invention, orof a pharmaceutical or veterinary composition according to the inventioncan be adjusted by the man skilled in the art according to the type andseverity of the infection (e.g. depending on the bacteria speciesinvolved in the disease, disorder and/or infection and its localizationin the patient's or subject's body), and to the patient or subject, inparticular its age, weight, sex, and general physical condition.

Particularly, the amount of vectors, delivery vehicles, bacteria,engineered bacteria, antibiotics, bacteriocins or endolysins accordingto the invention, preferably a payload according to the invention,particularly a payload packaged into a delivery vehicle according to theinvention, preferably a packaged plasmid or phagemid into a bacterialvirus particle according to the invention, or of a pharmaceutical orveterinary composition according to the invention, to be administeredhas to be determined by standard procedure well known by those ofordinary skills in the art. Physiological data of the patient or subject(e.g. age, size, and weight) and the routes of administration have to betaken into account to determine the appropriate dosage, so as atherapeutically effective amount will be administered to the patient orsubject.

For example, the total amount of delivery vehicles, particularly apayload packaged into a delivery vehicle according to the invention,preferably a plasmid or phagemid packaged into a bacterial virusparticle according to the invention, for each administration iscomprised between 10⁴ and 10¹⁵ delivery vehicles.

Definitions «Vector»

As used herein, the term “vector” refers to any construct of sequencesthat are capable of expression of a polypeptide in a given host cell. Ifa vector is used then the choice of vector is dependent upon the methodthat will be used to transform host bacteria as is well known to thoseskilled in the art. Vectors can include, without limitation, plasmidvectors and recombinant phage vectors, or any other vector known in thatart suitable for delivering a polypeptide of the invention to targetbacteria. The skilled artisan is well aware of the genetic elements thatmust be present on the vector in order to successfully transform, selectand propagate host cells comprising any of the isolated nucleotides ornucleic acid sequences of the invention.

«Delivery Vehicle»

As used herein, the term «delivery vehicle» refers to any vehicle thatallows the transfer of a payload into a bacterium.

There are several types of delivery vehicle encompassed by the presentinvention including, without limitation, bacteriophage scaffold, virusscaffold, bacterial virus particle, chemical based delivery vehicle(e.g., cyclodextrin, calcium phosphate, cationic polymers, cationicliposomes), protein-based or peptide-based delivery vehicle, lipid-baseddelivery vehicle, nanoparticle-based delivery vehicles,non-chemical-based delivery vehicles (e.g., transformation,electroporation, sonoporation, optical transfection), particle-baseddelivery vehicles (e.g., gene gun, magnetofection, impalefection,particle bombardment, cell-penetrating peptides) or donor bacteria(conjugation).

Any combination of delivery vehicles is also encompassed by the presentinvention.

The delivery vehicle can refer to a bacteriophage derived scaffold andcan be obtained from a natural, evolved or engineered capsid.

In some embodiment, the delivery vehicle is the payload as bacteria arenaturally competent to take up a payload from the environment on theirown.

In a particular embodiment, the delivery vehicle is a bacterial virusparticle, in particular a packaged phagemid.

In a particular embodiment, the delivery vehicle, in particular thebacteriophage, bacterial virus particle or packaged phagemid, comprisingthe vector of the invention is incapable of self-reproduction.

In the context of the present invention, “self-reproduction” isdifferent from “self-replication”, “self-replication” referring to thecapability of replicating a nucleic acid, whereas “self-reproduction”refers to the capability of having a progeny, in particular of producingnew delivery vehicles, said delivery vehicles being either producedempty or with a nucleic acid of interest packaged.

By “delivery vehicle incapable of self-reproduction” is meant hereinthat at least one, several or all functional gene(s) necessary toproduce said delivery vehicle is(are) absent from said delivery vehicle(and from said vector included in said delivery vehicle). In a preferredembodiment, said at least one, several or all functional gene(s)necessary to produce said delivery vehicle is(are) present in the donorcell as defined above, preferably in a plasmid or in a helper phagepresent in the donor cell as defined above, enabling the production ofsaid delivery vehicle in said donor cell.

In the context of the invention, said functional gene necessary toproduce delivery vehicle may be absent through (i) the absence of thecorresponding gene or (ii) the presence of the corresponding gene but ina non-functional form.

In an embodiment, the sequence of said gene necessary to produce saiddelivery vehicle is absent from said delivery vehicle. In a preferredembodiment, the sequence of said gene necessary to produce said deliveryvehicle has been replaced by a nucleic acid sequence of interest, inparticular by a nucleic acid sequence encoding enzymes or systems forinducing genetic modifications, as defined above.

Alternatively, said gene necessary to produce said delivery vehicle ispresent in said delivery vehicle in a non-functional form, for examplein a mutant non-functional form, or in a non-expressible form, forexample with deleted or mutated non-functional regulators. In apreferred embodiment, said gene necessary to produce said deliveryvehicle is present in said delivery vehicle in a mutated form whichrenders it non-functional in the target cell, while remaining functionalin the donor cell.

In the context of the invention, genes necessary to produce saiddelivery vehicle encompass any coding or non-coding nucleic acidrequired for the production of said delivery vehicle.

Examples of genes necessary to produce said delivery vehicle includegenes encoding phage structural proteins; phage genes involved in thecontrol of genetic expression; phage genes involved in transcriptionand/or translation regulation; phage genes involved in phage DNAreplication; phage genes involved in production of phage proteins; phagegenes involved in phage proteins folding; phage genes involved in phageDNA packaging; and phage genes encoding proteins involved in bacterialcell lysis.

«Payload»

As used herein, the term «payload» refers to any nucleic acid sequenceor amino acid sequence, or a combination of both (such as, withoutlimitation, peptide nucleic acid or peptide-oligonucleotide conjugate)transferred into a bacterium with a delivery vehicle.

The term «payload» may also refer to a plasmid, a vector or a cargo.

The payload can be a phagemid or phasmid obtained from natural, evolvedor engineered bacteriophage genome. The payload can also be composedonly in part of phagemid or phasmid obtained from natural, evolved orengineered bacteriophage genome.

In some embodiment, the payload is the delivery vehicle as bacteria arenaturally competent to take up a payload from the environment on theirown.

«Nucleic Acid»

As used herein, the term “nucleic acid” refers to a sequence of at leasttwo nucleotides covalently linked together which can be single-strandedor double-stranded or contains portion of both single-stranded anddouble-stranded sequence. Nucleic acids of the present invention can benaturally occurring, recombinant or synthetic. The nucleic acid can bein the form of a circular sequence or a linear sequence or a combinationof both forms. The nucleic acid can be DNA, both genomic or cDNA, or RNAor a combination of both. The nucleic acid may contain any combinationof deoxyribonucleotides and ribonucleotides, and any combination ofbases, including uracil, adenine, thymine, cytosine, guanine, inosine,xanthine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine andisoguanine. Other examples of modified bases that can be used in thepresent invention are detailed in Chemical Reviews 2016, 116 (20)12655-12687. The term “nucleic acid” also encompasses any nucleic acidanalogs which may contain other backbones comprising, withoutlimitation, phosphoramide, phosphorothioate, phosphorodithioate,O-methylphosphoroamidite linkage and/or deoxyribonucleotides andribonucleotides nucleic acids. Any combination of the above features ofa nucleic acid is also encompassed by the present invention.

“Phagemid” and “Packaged Phagemid”

As used herein the term “phagemid” or “phasmid” are equivalent and referto a recombinant DNA vector comprising at least one sequence of abacteriophage genome and which is preferably not able of producingprogeny, more particularly a vector that derives from both a plasmid anda bacteriophage genome. A phagemid of the disclosure comprises a phagepackaging site and optionally an origin of replication (ori), inparticular a bacterial and/or phage origin of replication. In oneembodiment, the phagemid does not comprise a bacterial origin ofreplication and thus cannot replicate by itself once injected into abacterium. Alternatively, the phagemid comprises a plasmid origin ofreplication, in particular a bacterial and/or phage origin ofreplication.

As used herein, the term “packaged phagemid” refers to a phagemid whichis encapsidated in a bacteriophage scaffold, bacterial virus particle orcapsid. Particularly, it refers to a bacteriophage scaffold, bacterialvirus particle or capsid devoid of a bacteriophage genome. The packagedphagemid may be produced with a helper phage strategy, well known fromthe man skilled in the art. The helper phage comprises all the genescoding for the structural and functional proteins that are indispensablefor the phagemid according to the invention to be encapsidated. Thepackaged phagemid may be produced with a satellite virus strategy, alsoknown from the man skilled in the art. Satellite virus are subviralagent and are composed of nucleic acid that depends on the co-infectionof a host cell with a helper virus for all the morphogenetic functions,whereas for all its episomal functions (integration and immunity,multicopy plasmid replication) the satellite is completely autonomousfrom the helper. In one embodiment, the satellite genes can encodeproteins that promote capsid size reduction of the helper phage, asdescribed for the P4 Sid protein that controls the P2 capsid size to fitits smaller genome.

«Peptide»

As used herein, the term “peptide” refers both to a short chain of atleast 2 amino acids linked between each other and to a part of, a subsetof, or a fragment of a protein which part, subset or fragment being notexpressed independently from the rest of the protein. In some instances,a peptide is a protein. In some other instances, a peptide is not aprotein and peptide only refers to a part, a subset or a fragment of aprotein. Preferably, the peptide is from 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 amino acids to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,25, 30, 40, 50, 100, 200 amino acids in size.

Example Materials and Methods Mice

MYH6-specific TCR transgenic mice (TCR-M) on the BALB/c background havebeen previously described (8). TCRM mice were maintained as heterozygousand transgene-negative littermates were used as controls. TCRM mice werere-derived to germ-free conditions by axenic 2-cell stage embryotransfer into pseudopregnant germ-free recipient females at the CleanMouse Facility, University of Berne, Switzerland, and bred andmaintained under germ-free conditions in flexible film isolators.Germ-free status was routinely monitored by culture-dependent (aerobicand anerobic) and -independent (Sytox DNA stain of cecal contents)methods and were additionally confirmed pathogen-free. In the indicatedexperiments, germ-free TCRM mice were transferred at 4 or 8 weeks of ageto the SPF facility in the Institute of Immunobiology, Kantonsspital St.Gallen and co-housed with SPF TCRM mice and transgene-negativelittermates. Rag1^(−/−) and DO11.10 mice were obtained from the Jacksonlaboratories. Homozygous OVA-TCR transgenic mice (DO11.10) were matedwith SPF BALB/c Thy1.1 mice from the TCRM colony of the St. Gallenfacility and offspring were used for analysis. Rag1^(−/−) mice wereco-housed with TCRM mice 3 weeks before set-up of the breeding pairs.All mice were on the BALB/c genetic background and were maintained inindividually ventilated cages. Experiments were performed in accordancewith federal and cantonal guidelines (Tierschutzgesetz) under permissionnumbers SG15/06, SG16/07, SG17/07 and BE55/14 following review andapproval by the respective Cantonal Veterinary Offices (St. Gallen andBerne, Switzerland).

Human Serum Samples and Clinical Data

Serum samples from patients enrolled in two different clinical studieswere analyzed: (i) “Cardiac β1-adrenoceptor autoantibodies in humanheart disease: rationale and design of the Etiology, Titre-Course, andSurvival” (ETiCS, AMITIS arm) (27), EC permission No 186/07 (Table 2)and (ii) “Dissecting the role of cross-reactive antimicrobial T and Bcell responses in myocarditis and heart failure—an exploratory researchproject Micro-DCM”, EC permission No 2017-01853 (Table 3 and Table 4).

TABLE 2 Demographic details and HLA-DQB1* alleles of ETiCS-AMITISmyocarditis patients and healthy control group¹ Diagnosis by Patient No.Age (years)² Sex³ HLA-DQB1* EMB⁴ 1 21.3 Male 03:01 03:01 Yes 2 32.8 Male03:01 06:02 Yes 3 58.3 Female 03:02 06:03 Yes 4 54.4 Female 02:02 06:04Yes 5 52.8 Male 03:01 03:01 Yes 6 50.1 Male 03:02 06:02 Yes 7 35.2 Male03:02 03:02 Yes 8 31.1 Male 02:01 03:01 Yes 9 64.7 Male 02:01 03:02 Yes10 42.0 Male 03:02 04:02 Yes 11 24.1 Male 03:02 03:03 Yes 12 21.4 Male02:02 03:01 Yes 13 23.8 Male 03:01 06:04 Yes 14 46.1 Male 03:01 03:03Yes 15 20.1 Male 03:03 06:02 Yes 16 28.9 Male 05:01 06:02 Yes 17 39.5Male 02:02 05:03 Yes 18 21.1 Male 04:02 06:02 Yes 19 25.3 Male 03:0106:84 Yes 20 27.7 Male 02:02 04:02 Yes 21 18.5 Male 05:01 06:03 Yes 2269.0 Male 03:01 05:03 Yes 23 18.6 Male 03:01 03:01 Yes 24 53.7 Male n.d.⁵ n.d. ⁵ Yes 25 32.0 Male n.d. ⁵ n.d. ⁵ Yes ¹Healthy controls sera wereobtained at the base line visit of the interventional study registeredas ISRCTN18360696. Female to male ratio of 1:0.38 (N = 18) and mean ageof 35 ± 8.3 years. ²Mean age of is 36.5 ± 15.5 years in ETiCS/AMITIS.³Female to male ratio of 1:11.5 in ETiCS/AMITIS. ⁴EMB: Endomyocardialbiopsy. ⁵ n.d. non-determined.

TABLE 3 Demographic details, HLA-DQB1* alleles and diagnosis ofMicro-DCM patients. Patient No. Age (years)¹ Sex² HLA-DQB1* Diagnosis³ 169.9 Female 03:01 03:01 Heart failure 2 23.6 Male 02:01 03:01Myocarditis 3 19.2 Male 03:02 06:03 Myocarditis 4 25.8 Male 03:01 03:02Myocarditis 5 23.9 Female 02:02 06:03 Myocarditis 6 40.9 Female 05:0106:03 Myocarditis 7 37.0 Male 05:01 06:03 Myocarditis 8 54.9 Male 02:0104:02 Heart failure 9 63.5 Male 02:01 02:02 Myocarditis 10 65.0 Male03:02 06:02 Myocarditis 11 42.8 Male 02:01 03:03 Myocarditis 12 47.7Male 02:01 06:03 Heart failure 13 20.0 Male 03:03 06:02 Myocarditis 1439.1 Female 03:03 05:03 Myocarditis 15 26.0 Male 04:02 05:01 Myocarditis16 51.2 Male 02:02 05:03 Heart failure 17 30.5 Male 03:01 05:03Myocarditis ¹Mean age in myocarditis patients is 35.8 ± 15.2 and 55.9 ±9.7 years for heart failure patients. ²Female to male ratio formyocarditis patients of 1:3.3 and 1:3 for heart failure patients.³Myocarditis: patients with typical late enhancement pattern in cardiacMRI and exclusion of coronary artery disease by angiography or computedtomography. Heart failure: patients with left ventricular ejectionfraction ≤40%, no obvious mechanism underlying left ventriculardysfunction and no significant coronary artery by invasive angiography(no stenosis >50% in any main vessel).

TABLE 4 Demographic details and HLA-DQB1* alleles of healthy volunteers.Volunteer No. Age (years)¹ Sex² HLA-DQB1* 1 31.5 Female 02:01 03:03 240.3 Male 03:01 05:03 3 33.2 Male 03:01 05:01 4 55.4 Male 03:01 03:01 527.7 Female 06:04 06:02 6 35.1 Male 03:01 05:01 7 26.7 Female 02:0203:19 8 31.8 Male 03:01 05:01 9 31.1 Male 03:03 05:02 10 41.2 Female03:05 03:01 11 46.5 Female 03:02 05:02 12 32.8 Female 02:02 06:04 ¹Meanage 36.1 ± 8.3 years. ²Female to male ratio 1:1.

All study Participants provided written informed consent in accordancewith the Declaration of Helsinki and the International Conference onHarmonization Guidelines for Good Clinical Practice. All regulationswere followed according to the German or Swiss authorities and accordingto the clinical protocols. The retrospective part of this study (AMITIScohort) is a secondary investigation using patient samples collectedfrom existing clinical trials. Therefore, the sample sizes in thisreport were determined by the original clinical trial designs and sampleavailability; no additional inclusion/exclusion criteria were applied.The sample size calculation for the Micro-DCM study has not been donebecause of the exploratory nature of the study design.

Histology and Myocarditis Scoring

Histological analysis was performed as previously described (8).Briefly, hearts were fixed in 4% formaldehyde (Formafix) for at least 12h and embedded in paraffin. Histopathological changes were evaluatedfollowing hematoxylin/eosin (HE) and Elastica van Gieson (EVG) staining.Myocarditis severity was evaluated using a semiquantitative scoringsystem: 0, no inflammation; 1, <100 inflammatory cells involved, smallinflammatory lesions; 2, >100 inflammatory cells involved, largerinflammatory lesions; 3, >10% of the heart section involved ininflammation; 4, >30% of the heart section involved in inflammation;5, >30% of the heart section involved in inflammation with extensivefibrosis and dilation of ventricle. Images from heart sections wereacquired using a Leica DMRA microscope.

Flow Cytometry and Cell Isolation

For the generation of single-cell suspensions, spleens and lymph nodeswere collected in BSS and were mechanically disrupted on a metallicgrid. To confirm the presence of heart-infiltrating cells, mice received2 μg of anti-CD45.2 BV510 or anti-Thy 1.1 PE i.v. 5 min before beingeuthanized in the indicated experiments. Euthanized animals wereperfused with 20 ml PBS and small heart tissue pieces were placed into a6-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM HEPES(Lonza), 1 mg/ml collagenase D (Sigma) and 25 μg/ml DNasel (Applichem)and incubated at 37° C. under continuous stirring. The remaining tissuepieces were mechanically disrupted and mononuclear cells were purifiedby centrifugation (25 min at 800×g, 4° C.) on a 30-70% Percoll gradient(GE Healthcare). For isolation of colonic lamina propria cells, colonswere flushed and tissue was harvested and incubated three times for 15min at room temperature under constant agitation with BSS containing 5%FCS (Lonza), 5 mM EDTA (Sigma), 10 mM HEPES (Sigma) and 1 mM DTT inorder to dissociate the epithelial layer. The tissue was subsequentlywashed with BSS containing 10 mM HEPES and digested three times for 20min at 37° C. with 120 μg/ml collagenase P (Roche), 25 μg/ml DNAse I(Applichem) and 5 μg/ml Dispase I (Roche) in RPMI-1640; after obtaininga single cell suspension, cells were purified by a 30-70% Percollgradient. Single-cell suspensions were first stained with the fixableviability dye Zombie Aqua (Biolegend) and incubated for 30 min on ice;after washing, cells were incubated for 20 min at 4° C. in PBScontaining 2% FCS and 10 mM EDTA with fluorochrome-labeled antibodies(Table 5). Cells were acquired with a BD LSRFortessa (BD Biosciences)and analyzed using FlowJo software (Treestar Inc) following stablishedguidelines (32).

TABLE 5 Antibodies used in this study Clone Reagent Conjugate DilutionSource IA8 anti-Ly6G PE 1:200 Pharmigen AL-21 anti-Ly6C PerCP 1:100Biolegend 145-2C11 anti-CD3e PerCP 1:100 Biolegend 30-F11 anti-CD45APC-Cy7 1:100 Biolegend 30-F11 anti-CD45 PE 1:100 Biolegend IM7anti-CD44 APC-Cy7 1:100 Biolegend MEL-14 anti-CD62L BV-421 1:100Biolegend M1/70 anti-CD11b BV-711 1:100 Biolegend FJK16S anti-FoxP3PeCy7 1:100 eBioscience B20.1 anti-Vα2 APC 1:100 BDBioscience MR5-2anti-Vβ8 FITC 1:100 BDBioscience RM4-5 anti-CD4 BV-605 1:100 BiolegendXMG-1.2 anti-IFN © APC 1:100 Biolegend TC11- anti-IL17 PE 1:100Biolegend 18H10.1 KJ1-26 anti-TCR D011.10 FITC 1:100 Biolegend 2G9anti-IA/IE FITC 1:100 BDBioscience SA011F11 anti-CX3CR1 APC- 1:100Biolegend Fire750 2B10C42 anti-MERTK APC 1:100 Biolegend 104 anti-CD45.2BV-605 1:100 Biolegend SA203G11 anti-CCR2 BV-421 1:100 BiolegendX54-5/7.1 anti-CD64 PeCy7 1:250 Biolegend DATK32 anti-α4β7 APC 1:100Biolegend 29-2L17 anti-CCR6 PE 1:100 Biolegend 11-44-2 anti-IgA PE 1:50 eBiosciences

Immunofluorescence

Murine colonic tissue and lymph nodes were fixed overnight at 4° C. infreshly prepared 4% paraformaldehyde (Merck Millipore) under agitation.Tissues were embedded and oriented in 4% low melting agarose(Invitrogen) in PBS and serially sectioned with a vibratome (LeicaVT-1200). 30-40 μm thick sections were blocked in PBS containing 10%FCS, 1 mg/ml anti-Fcγ receptor (BD Biosciences) and 0.1% Triton X-100(Sigma). Tissues were incubated overnight at 4° C. with the followingantibodies: anti-mouse CD4, anti-mouse E-Cadherin (Biolegend).Microscopy was performed using a confocal microscope (LSM-710, CarlZeiss). Microscopy data were recorded with ZEN 2010 software (CarlZeiss, Inc.) and processed in Imaris Versions 7 (Bitplane).

Ex Vivo Restimulation and Cytokine Production of Murine T Cells

For assessment of ex vivo production of IFN-γ and IL-17, 10⁶ lymphocyteswere incubated for 3.5 h at 37° C. in 96-well round-bottom plates in 200μL of RPMI per 5% FCS supplemented with 10 μg/mL brefeldin A (Sigma).Cells were stimulated with 0.25 μg of the different peptides (seebelow), or phorbol myristate acetate (50 ng/ml; Sigma)/ionomycin (500ng/ml; Sigma) (PMA/I) as positive control, or were left untreated. Aftersurface molecule labelling, cells were stained with the fixableviability dye Zombie Aqua (Biolegend). Cells were fixed withcytofix-cytoperm (BD Biosciences) for 20 min. Fixed cells were incubatedat 4° C. for 40 min with permeabilization buffer (2% FCS, 0.5% saponinin PBS) containing antibodies to CD3, CD4, IFN-γ and IL-17A. Sampleswere measured using a BD LSR Fortessa (BD Biosciences). Data wereanalyzed using FlowJo software (Tree Star, Inc.).

Generation of B. thetaiotaomicron ΔBT1626 Mutant Strain

Deletion of the B. thetaiotaomicron β-galactosidase (BT1626) was doneusing a counterselection system described in (33). A strain with a cleandeletion of tdk (BT2275) in the VPI-5482 background, resistant to thetoxic nucleotide analog 5-fluoro-2′-deoxyuridine (5FUdR) was used as astarting point. Genomic regions 1 kb upstream and downstream of BT1626,respectively, were amplified via colony per, with primers includingoverhangs for Gibson assembly into the plasmid pExchange-tdk (33),digested with the enzymes XbaI and SaII. All primers are listed in Table6.

TABLE 6 Primers used for the deletion of B. thetaiotaomicron BT1626 NameSequence Purpose Bt_bgal_ko_1 AAG ATA ACA TTC GAG TOGForward primer 1 kb upstream ACT ATT TCG GCA ATA ATAof BT1626, with Gibson CGC TGA AAG (SEQ ID NO: overhang homologous to 3)pExchange-tdk. Bt_bgal_ko_2 CAA ATG CAA TCA GTT TCAReverse primer immediately GGC ATT ATT ATA TTG TTTupstream of BT1626, with TTT GGT GAC TGG T (SEQGibson overhang homologous ID NO: 4) to the downstream region of BT1626.Bt_bgal_ko_3 CCT GAA ACT GAT TGC ATT Forward primer immediatelyTGG (SEQ ID NO: 5) downstream of BT1626 Bt_bgal_ko_4 GCG GTG GCG GCC GCTReverse primer 1 kb CTA GAA GTC GTT TGT TGT downstream of BT1626, withTTT CTT CG (SEQ ID NO: 6) Gibson overhang homologous to pExchange-tdk.1626_250us_f CAC ATA GTA GTT TTC TTT Control primer 250 bpCGT C (SEQ ID NO: 7) upstream of BT1626. 1626_264inside_rGCG AAA CTG ACG GAT Control primer 264 bp inside CAG (SEQ ID NO: 8)the BT1626 coding sequence. 1626_250ds_r TCG GAC GAT AAT GCGControl primer 250 bp ACT T (SEQ ID NO: 9) downstream of BT1626.

The plasmid was then transformed into the conjugative E. coli strainSM10 and conjugated into the B. thetaiotaomicron Δtdk. The resultingcolonies are resistant to erythromycin and have the complete plasmidinserted into the chromosome resulting from a single homologousrecombination event. Counterselection on plates containing 200 μg/ml5FUdR led to a second recombination event, and either to a reversion tothe wild type state or to the deletion of BT1626. Successful deletionwas tested using PCR with control primers, and clones with a cleandeletion of the gene were further used (FIG. 7F and Table 6). All platesused for generating mutants were Brain Heart Infusion (Oxoid),supplemented with 2% agar (Sigma), 10% defibrinated sheep blood (Sigma),1 μg/ml Hemin (Sigma). 200 μg/ml FUdR (Sigma), 25 μg/ml erythromycin(Sigma), and 200 μg/ml gentamicin (Sigma) were added before pouring theplates when needed.

Bacterial Culture and Monocolonized Mice

The different Bacteroides thetaiotaomicron strains (ATCC29148,ATCC29148^(Δtdk) or ATCC29148^(Δtdk)ΔBT1626) were grown anaerobicallyovernight at 37° C. in Brain Heart Infusion (BHI, CM1135, Oxoid) withthe addition of hemin (1 μg/ml, Sigma) and menadione (0.5 μg/ml, Sigma).E. coli BL21(DE3) was grown in LB medium at 37° C. Overnight brothculture of B. thetaiotaomicron or E. coli was washed once in PBS andgavaged intra-gastrically (500 μl/mouse corresponding to 10⁹bacteria/mouse) to germ-free mice (at least 3-4 week old). Germ-free andmonocolonized animals were kept in flexible film isolators until the endof the experiments and monitored regularly for excluding anycontaminations using Sytox staining on fecal pellets for GF animals,Gram staining and 16S rRNA sequencing on fecal pellets for monocolonizedanimals (34). The effects of mono-colonization in TCRM mice wereanalyzed 8 weeks post colonization.

Myocarditis Induction in Rag1^(−/−) Mice

Spleens were collected from TCRM mice and disrupted on a 70-μm cellstrainer. Red blood cells were lysed by osmotic shock and 10⁶splenocytes were injected intravenously in 4 wk mice in the lateral tailvein of Rag1^(−/−) mice. Seven days after adoptive transfer, mice werebled to confirm CD4⁺ T cell expansion. Disease activity scores andanalysis of T cell activation in the heart and colonic lamina propriawere performed at day 28 post adoptive transfer.

Antibiotics Treatment

Mice were treated for 4 or 16 weeks with the following antibiotics inthe drinking water (i) a broad spectrum antibiotic mixture; Sulfadoxine400 mg/l and Trimethoprim 80 mg/l (Borgal; Veterinaria) or (ii)Streptomycin 200 mg/l (Sigma-Aldrich) to reduce the proportion ofBacteroidetes (35) or iii) Vancomycin 100 mg/l. Metronidazole(Sigma-Aldrich) was administered by gavage at a dose of 2 mg every 3days to Sulfadoxine and Trimethoprim-treated mice or a separate group ofmice for exclusive Metronidazole treatment.

Ex Vivo Proliferation Assay and Peptides Used in the Study

Spleen cells were labelled with 10 μl of 5 mM carboxyfluoresceinsuccinimidyl ester (CSFE, dissolved in DMSO) (Molecular Probes) in 10 mlof PBS for 10 min at 37° C. The staining reaction was stopped with 1 mLFCS (Lonza), followed by washing with PBS. A total of 2×10⁵splenocytes/well were seeded in 96-well round-bottom plate andMYH6₆₁₄₋₆₂₉ (RSLKLMATLFSTYASADR, SEQ ID NO: 10) or Bacteroidesβ-galactosidase₁₁₋₂₅ (TFLILMAALTATFASAQK, SEQ ID NO: 11), Enterobactercysteine hydrolase₁₀₋₂₇ (LLIGMMSTFSTYASAQET, SEQ ID NO: 12) orOVA₃₂₃₋₃₃₉ (ISQAVHAAHAEINEAGR, SEQ ID NO: 13) peptide was added at theindicated concentrations. CSFE dilution was assessed by flow cytometryafter 3 days of incubation at 37° C. All peptides were synthetized byGenscript with >80% of purity.

In Vivo Proliferation of TCRM Cells

Spleen cells were labelled with CFSE as described above and 5×10⁶ cellswere i.v. transferred in to Rag^(−/−) mice. Mice were culled at days 2,3, 4, or 7 after adoptive transfer and the presence of myosin specific(CD4⁺Vα2⁺Vβ8⁺) cells as well as CFSE dilution was assessed by flowcytometry in single cell suspensions from the mediastinal lymph node,colonic lymph node, colonic lamina propria and heart.

Detection of Bacteria-Specific IgG

High-binding 96-well polystyrene plates (Corning) were coated with 10⁷CFU of the following bacterial strains B. theta ATCC29148, B.distasionis, B. vulgatus, E. cloacae ATCC 13047 or E. coli K12 in 0.1 Mcarbonate-bicarbonate buffer, pH 9.5. Plates were incubated for 1 h at37° C. and then overnight at 4° C. Before use, plates were washed threetimes with PBS containing 0.05% Tween-20 (PBS-T) (Sigma-Aldrich).Non-specific binding was blocked with 5% non-fat dry milk diluted in PBS(PBS-M) for 1 h at 37° C. After washing, sera were diluted 1:20 in PBS-Mand two fold serial dilutions were added to the wells. Plates wereincubated for 1 h at 37° C., followed by four washes with PBS-T and thenincubated for 1 h of at 37° C. with peroxidase-conjugated rabbitanti-human IgG (1:2500) or rabbit-anti-mouse IgG (1:1000) antibody (inPBS-M, Jackson Immuno Research). After four washes with PBS-T,ortho-phenylenediamine (0.5 mg/ml; Sigma) in 0.1 M citrate buffer, pH5.6, containing 0.08% H₂O₂ was used to develop the reaction. Opticaldensity was measured at 492 nm using an automated ELISA plate reader(Tecan). Absorbance values at 1:160 dilutions are presented in thefigures. In human samples high and low responders to B. thetaiotaomicronwere high responders were defined as patients that had an IgG atdiagnosis of A >mean of healthy controls +2 SD.

Bacterial IgA Binding Detected by Flow Cytometry

Fecal-bound IgA was assessed using bacterial flow cytometry forIgA-bound bacteria performed as previously described (36). In brief,fecal pellets were homogenized in 1 ml of staining buffer (PBS+1% (w/v)bovine serum albumin (BSA, Sigma). Tubes were centrifuged at 400 g for 5minutes to pellet large debris and the supernatant was filtered througha sterile 70 μm cell strainer and transferred to a new tube. Sampleswere centrifuged at 8000×g for 5 minutes to pellet bacteria and pelletswere re-suspended in PBS with SYTO BC (1:1000, Life Technologies) for 15minutes on ice, Samples were washed with PBS and stained in 100 μlstaining buffer containing PE-conjugated anti-mouse IgA (1:75) for 30minutes on ice. Samples were washed 3 times with PBS and re-suspendedfor flow cytometry analysis in a FACS CANTO (BD). Bacteria were gatedbased on forward and side scatter and the gating strategy was verifiedby SYTO-BC staining. Fecal samples from Rag1^(−/−) mice were used asnegative controls to define the threshold for IgA⁻ and IgA⁺ populations.Analysis of IgA binding was done using FlowJo (v10.1).

Microblota Analysis

For microbial composition analysis, the feces from TCRM SPF mice, Tg⁻littermates and TCRM GF co-housed with TCRM SPF and Tg⁻ littermates wereused. Microbial composition was assessed by a 16S high-throughputamplicon analysis as described previously (37). The 163 rRNA genesegments spanning the variable V5 and V6 regions were amplified from DNAfrom the samples, using a multiplex approach with the barcoded forwardfusion primer 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ ID NO:14) BARCODEATTAGATACCCYGGTAGTCC-3′ (SEQ ID NO: 23) in combination with the reversefusion primer 5′-CCTCTCTATGGGCAGTCGGTGATACGAGCTGACGACARCCATG-3′ (SEQ IDNO: 15). The PCR-amplified 16S V5-V6 amplicons were purified andprepared for sequencing on the Ion torrent PGM system according to themanufacturers instructions (Life Technologies). Samples with over 10′000reads were accepted for analysis. Data analysis was performed using theQIIME pipeline version 1.8.0 (38). Operational taxonomic units werepicked using UCLUST with a 97% sequence identity threshold, followed bytaxonomy assignment using either the latest Greengenes database. Forestimation of species diversity within samples (α-diversity), theShannon index was calculated using the R phyloseq package afterrarefaction to even depth to the sample with the lowest number ofsequences (34).

Fecal Bacterial DNA Extraction and Quantitative B. thetaiotaomicron PCR

Bacterial genomic DNA was extracted using a commercial kit (QIAamp DNAStool Mini Kit) with minor modifications. Glass beads (Sigma, G4649-10G)were used for an optimal disruption of fecal content and an additionalextraction step was introduced after incubation of bacteria with a lysisbuffer (20 mg/ml lysozyme—Sigma 62970-5G-F, 2 mM EDTA, 1.2% Triton, 20mM Tris-HCI). Extracted genomic DNA was used as a template for B.thetaiotaomicron PCR amplification and quantification using a real-timePCR machine (Quant Studio 5) and SYBR Green PCR technology (AppliedBiosystems, Thermo Fisher Scientific). B. thetaiotaomicron wasquantified using species specific primers that have been established andvalidated before (39) (atcgcaaaaataagatgggcaaa andcacaacagccatagcgttcca) with a cycling protocol of denaturation at 95° C.(2 min), 40 cycles of 95° C. (20 sec), 58° C. (20 sec) and 72° C. (30sec), Each 17 μl qPCR mixture consisted of 8.5 μl of 2× SYBR GreenMasterMix (Applied Biosystems, Thermo Fisher Scientific), 1.7 μl of BSA(100 μg/ml), 0.6 μl of each primer (10 μM), 3.60 μl PCR-grade water and2 μl of extracted genomic DNA. DNA extracted from B. theta ATCC29148 wasused to construct a standard curve with 10-fold dilutions of DNAtemplates of known concentrations. Concentrations of DNA used in thestandard curves ranged from 33.75 ng/μl equivalent to 10⁷ bacteria to0.035 pg/μl equivalent to 10¹ bacteria. The PCR amplifications wereperformed in duplicates and the detection limit was 0.34 pg/μl (10²bacteria). Bacterial qPCR signals were normalized to 1 μg of totalbacterial DNA.

Bioinformatics Analysis of Peptide Homology and MHC Class II Binding

Proteins encoded in cultured and uncultured bacteria of the NCBImicrobial protein database were searched for 15 amino acid sequencessimilar to MYH6₆₁₄₋₆₂₉ with BLAST p2.3.1 (blast.ncbi.nlm.nih.gov).Predicted binding of the candidate bacterial peptides to the BALB/cmouse IA^(d) MHC class II was evaluated using the immune epitopedatabase and analysis resource IEDB from the National Institute ofAllergy and Infectious diseases online prediction tool(tools.iedb.org/mhcii/). The binding threshold was set at 10, andpeptides with percentile ranks lower than the threshold value werepredicted to be good MHC II haplotype binders (Table 1).

TABLE 1Sequences and properties of microbial peptide mimics of MYH6₆₁₄₋₆₂₉.Protein Ia^(d) Murine length binding MYH6 Accession (amino Amino acid(percent- identity Description Organism number acids) sequence² ile)³(%) Myosin heavy Mus musculus NP_034986.1 1938 SLKLMATLFSTYASAD  2.12chain 6 (SEQ ID NO: 16) (MYH6) Myosin heavy Homo sapiens NP_002462.21939 SLKLMATLFSSYATAD  8.39 87.50 chain 6 (SEQ ID NO: 17) (MYH6) β-Bacteroides WP_109116112.1 1022 FLILMAALTATFASAQ  2.71 56.25galactosidase faecis ¹ (SEQ ID NO: 18) β- Bacteroides SQA30530.1 1023FLILMAALTATFASAQ  2.71 56.25 galactosidase thetaiotaomicron ¹(SEQ ID NO: 19) Cysteine Enterobacter KJX05885.1  238 LIGMMSTESTYASAQE16.78 50.00 hydrolase cloacae ¹ (SEQ ID NO: 20) MFS EnterococcusAWM66597.1  475 GMTLMGLSTLFLSTYA 14.00 37.50 transporter faecalis ¹(SEQ ID NO: 21) Outer Chlamydia CAA39396.1  547 LETSMAEFTSTNVISL 21.8725.00 membrane pneumoniae (SEQ ID NO: 22) protein 2 ¹Commensal in humanintestina microbiome. ²Amino acids matching to murine MYH6₆₁₄₋₆₂₈ arehighlighted by underlining. ³MHC class II Ia^(d) binding predictionalgorithm: IEDB analysis resource (low percentile rank indicates betterbinding to MHC II molecule).

The predicted binding of human MYH6₆₁₄₋₆₂₉ and Bacteroides β-gal₁₁₋₂₅peptides to HLA-DQ alleles was performed using the full HLA referenceset, which provides >99% of coverage of the HLA allele usage (40). Inputsequences from full human myosin heavy chain alpha (myosin heavy chain6, MYH6) protein (accession number: NP_002462.2) and B. thetaiotaomicronβ-galactosidase (accession number: SQA30530.1) were used for theprediction in IEDB.

HLA Analysis of Myocarditis Patients

The HLA-genotyping for the AMITIS patients was performed by the Institutfür Klinische Transfusionsmedizin and Hämotherapie from theUniversitätsklinikum Wurzburg. Briefly, genomic DNA was obtained fromPBMCs and analyzed by a sequence based typing (SBT) by Sanger sequencingusing forward and reverse primers for sequencing the exons 2 and 3 (HLAclass H) in a Applied Biosystems Genetic analyzer 3130×I, instrument(ThermoFisher Scientific), using SBTexcellerator kits (Gendx) and theanalysis software SBTengine. The Micro-DCM cohort HLA typing wasperformed by Illumina sequencing (Histogenetics USA).

Detection of Anti-β1 Antibodies in the Sera of Patients

To detect anti-β1 antibodies in the sera of patients we used Dynabeads®M-270 Epoxy (Life Technologies) coated for 72 h according to themanufacturer's instructions with 100, 80, 60, 40, 20, 10, 5 or 2.5 μg/mlβ1-ECII peptide. Control beads were coated with 10 μg/ml of a scramblepeptide comprising the same amino acids of the β1-ECII peptide. Afterthe coating the beads were washed using phosphate-buffered saline(PBS)/0.1% Bovine Serum Albumin (BSA) and stored in PBS/0.1% BSA/0.02%NaN3 until further use. For antibody detection 10⁴ beads of everyfraction were seeded into a 96-well-V-bottom plate. After blocking thebeads with phosphate-buffered saline (PBS)/10% Bovine Serum Albumin(BSA) (15 min, on ice), a 1:100 dilution in PBS/0.1% BSA/0.02% NaN3 ofpatient serum, or as negative control pooled human AB serum (Sigma), wasincubated with the beads in a total volume of 50 μl (15 min, on ice). Asa positive control the rat anti-β1-ECII monoclonal antibody 13F6 wasused (10 μg/ml in PBS/0.1% BSA/0.02% NaN3/AB serum 1:100). The beadswere washed four times using PBS/0.1% BSA/0.02% NaN3 and transferred tofresh wells before incubation with a 1:100 dilution of the secondaryantibody F(ab)2 donkey-anti-human IgG (H+L) RTC (Dianova) in PBS/0.1%BSA/0.02% NaN3 (total volume: 50 μl, 15 min, on ice). 13F6 was detectedusing F(ab)2 donkey-anti-rat IgG (H+L) PE (Dianova; 1:1000 dilution inPBS/0.1% BSA/0.02% NaN3). After a final washing step the samples weremeasured on a FACScan flow cytometer and the data analyzed using FlowJo(Treestar). Half maximal binding was determined using GraphPad Prism.When a half maximal binding value was calculable, the serum was definedas antibody-positive.

Human T Cell Stimulation

IFN-γ ELISPOT was performed as described by Lv et. al. with minormodifications (7). Briefly, PBMCs from patients and healthy volunteerswere isolated on density gradients. PBMCs were adjusted to 2×10⁶cells/ml in 0.5 ml of complete IVS medium supplemented with 8% of humanserum. Cells were stimulated for 48 h with 0.25 μg/ml of the indicatedpeptide or left untreated as medium control. After incubation, cellswere washed and re-suspended in 0.3 ml of RPMI/10% FCS and approximately5×10⁵ cells/0.1 ml were dispensed in duplicates into previously coatedand blocked ELISPOT plates (IFN-γ base kit, MABTECH). After 24 h ofincubation at 37° C. the ELISPOT plates were washed and developed asindicated by the manufacturer. Plates were counted using an ELISPOTreader and analyzed with the software ELISPOT 3.1SR (AID). Number ofspecific IFN-γ positive cells was calculated as the mean of theduplicates minus the mean of the medium control.

Human Fecal Microbiota Transplantation

Fecal microbiota transplantation was performed as previously reported byGopalakrishnan et. al (41). Briefly, germ free TCRM andtransgene-negative littermates received FMT from myocarditis patientswhich were previously confirmed to be positive for B. thetaiotaomicronand the DQB1*03:02 HLA allele; each patient sample was administered to 4mice. 200 μl cleared supernatant from 0.1 g/μl human fecal suspensionwas obtained using a 100 μm strainer and gavaged into mice 3 times over1 week. B. thetaiotaomicron colonization was assessed by qPCR at 14 and21 days after the first administration.

Statistical Analysis

Statistical analyses were performed with Graphpad Prism 7.0 usingunpaired two-tailed Student's t-test or Mann-Whitney U tests.Longitudinal comparison between different groups was performed byone-way ANOVA with Tukey's post-test or two-way ANOVA with Bonferroni'spost-test. For allele comparison, the Fisher's exact test was used.Statistical significance was defined as p<0.05.

Results Microbiome-Dependent Inflammatory Cardiomyopathy

The link between infection with pathogens and the development ofautoimmune diseases is well-documented (17, 18), while the impact ofcommensal bacteria on dysregulated self-reactivity has only recentlybeen uncovered (19-23). Importantly, commensal bacteria such asBacteroides can also dampen autoimmune reactivity both in mice andhumans (24, 25). To assess whether the intestinal environment and thevast repertoire of antigens present in the microbiota shapeheart-specific autoimmunity, we used transgenic mice that express aMYH6-specific T cell receptor on more than 95% of their CD4⁺ T cells(TCRM) (8). As described previously (8), all TCRM mice developedspontaneous autoimmune myocarditis and approximately 50% of the animalsprogressed from autoimmune myocarditis to lethal DCM under specificpathogen free (SPF) housing conditions (FIG. 1A-D). In stark contrast,the lack of a commensal microbiome under germ-free (GF) conditionsrescued TCRM mice from progression to the lethal disease (FIG. 1A),abrogated cardiac dilatation (FIG. 1B) and substantially reducedfibrotic remodeling of the cardiac tissue in 12 week old animals (FIG.1C). Histopathological assessment of disease severity revealed thatmyocarditis in GF TCRM mice was significantly reduced at 12 weeks whencompared to age-matched TCRM mice raised under SPF conditions (FIG. 1D).Echocardiographic analysis of heart functions with recording of theejection fraction (FIG. 1E), fractional shortening (FIG. 1F) andsystolic left ventricle internal diameter (LVID) (FIG. 1G) revealed thathearts of 12 week old GF TCRM mice functioned normally despite the lowlevel cardiac inflammation. The small difference in disease severity at4 weeks of age and the lack of progressive myocarditis in GF TCRM mice(FIG. 1D) suggested that post-weaning processes such as microbialcolonization critically impact disease aggravation in TCRM mice.

To determine to which extent microbial colonization affected theactivation of heart-specific CD4⁺ T cells, TCRM mice were transferredfrom GF to the SPF environment at 4 and 8 weeks of age and co-housed(CoH) with SPF TCRM mice (FIG. 1H). Colonization with the SPF microbiomeprecipitated progression to lethal disease in ex-GF mice within 6-12weeks (FIG. 1I), significantly exacerbated cardiac inflammation (FIG.1J) and impaired cardiac function (FIG. 5A-C). Moreover, microbialcolonization of GF TCRM mice led to rapid infiltration of the heart withCD45⁺ immune cells (FIG. 1K) and an overshooting accumulation ofMYH6-specific CD4⁺ T cells that expressed the transgenic Vβ8 chain (FIG.1L). While IFN-γ production of heart-infiltrating TCRM Th cells did notdiffer between SPF and GF mice (FIG. 1M), the expression of IL-17 wasdependent on the microbial status (FIG. 1N). Co-housing increased thefraction of both IFN-γ-(FIG. 1M) and IL-17-secreting heart-specific CD4⁺T cells (FIG. 1N) and promoted the accumulation of inflammatory myeloidcells in the heart tissue (FIG. 1O-R and FIG. 5D-F) includinginflammatory monocytes expressing CCR2 (FIG. 1P), MHCII^(high)macrophages (FIG. 1Q) and MERTK⁺ macrophages (FIG. 1R). These datasuggest that the presence of microbiota fosters the imprinting of a Th17phenotype in heart-specific CD4⁺ T cells and favors the accumulation ofinflammatory monocytes/macrophages in the myocardium of TCRM mice.

Intestinal Peptide Mimics Activate Heart-Specific Th Cells

IL-17 determines the severity of experimental myocarditis in mice (6, 8)and appears to promote heart failure in human myocarditis patients (9).Since balancing of Th17 cell activity is crucial for the integrity andimmune homeostasis of the intestinal lamina propria (26), wehypothesized that MYH6-specific CD4⁺ T cells in TCRM mice receive remoteactivation signals from the intestinal microbiota. Indeed, assessment ofmucosal homing molecule expression on heart-infiltrating CD4⁺ TCRM cellsrevealed significantly elevated integrin α4β7 and chemokine receptorCCR6 expression under SPF compared to GF conditions (FIG. 2A). Adoptivetransfer of dye-labeled TCRM cells into Rag1-deficient BALB/c mice (FIG.6A) revealed homing of CD4⁺ cells to the T cell zones of colonic patches(FIG. 2B), colonic lymph nodes (FIG. 6B) and mesenteric lymph nodes(FIG. 6C). The adoptively transferred Vβ8⁺ CD4⁺ T cells showeddetectable proliferation on day 3 in the lamina propria, the coloniclymph node and the mediastinal lymph node, while heart-infiltrating TCRMcells could only be detected on day 7 (FIG. 2C and FIG. 6D). The CD4⁺ Tcell response in the hearts of 4 week old TCRM mice was dominated byIFN-γ-producing Th cells (FIG. 6E), whereas the colonic lamina propriaenvironment fostered the differentiation of IL-17-producing CD4⁺ T cells(FIG. 6E and F). Importantly, only MYH6-specific CD4⁺ T cells, but notovalbumin-specific CD4⁺ T cells from the colonic lamina propria wereable to secrete IFN-γ or IL-17 upon ex-vivo re-stimulation with the MYH6peptide (FIG. 6G). These data indicate that heart-specific Th cells canbe activated in the colonic lamina propria and/or the associatedlymphoid organs prior to receiving further antigenic stimulation in theheart.

To further explore the interaction of heart-specific Th cells and theintestinal microbiome, 4 week old GF TCRM mice were co-housed with SPFtransgene-negative (Tg⁻) and SPF TCRM littermates (FIG. 2D). Nextgeneration sequencing of 16S rRNA genes amplified from feces revealedthat co-housed Tg⁻ and TCRM mice possess disparate microbiomes (FIG. 6H)and that the microbiome of co-housed ex-GF TCRM mice appears to be moresimilar to the SPF TCRM microbiome (FIG. 2E). More detailed analysesconfirmed that GF TCRM mice acquired the microbiome of SPF TCRM mice(FIG. 2F) with particularly high changes in the genera Bacteroides andParabacteroides (FIG. 6I). These data suggested that MYH6-specific CD4⁺T cells in the TCRM mice impact the composition of the microbiomethrough antigen-specific interactions. Indeed, an in silico search forpotentially cross-reactive epitopes identified two mimic peptides withhigh similarity to MYH6 in the β-galactosidase (β-gal) of Bacteroidesthetaiotaomicron (B. theta) and B. faecis that possesses a homology of56%, and cysteine hydrolase peptide of Enterobacter cloacae with 50%homology (Table 1). The B. theta β-gal peptide but not the Enterobactercloacae peptide, induced in vitro proliferation of CD4⁺ T cells fromTCRM mice (FIG. 2G), while ovalbumin-specific CD4⁺ T cells were notactivated by the microbial peptides (FIG. 7A). TCRM CD4⁺ T cells exhibita functional avidity of 6.3×10⁻⁶ M for the cross-reactive B. thetapeptide (FIG. 7B) that facilitated efficient activation of TCRM T cellsin vivo by B. theta peptide-pulsed dendritic cells (FIG. 6C) and ex vivore-stimulation of transgenic CD4⁺ T cells from heart and colon (FIG. 2H)of 12 week old TCRM mice. To elucidate the role of B. theta for theactivation of TCRM Th cells in the colonic lamina propria, wemono-colonized GF TCRM mice with B. theta or Escherichia (E.). coli, orcolonized them with our SPF microbiota (FIG. 7D). These analyses showedthat only B. theta mono-colonization specifically fostered the inductionof IL-17 production as seen following SPF colonization (FIG. 7E). Thedifferentiation towards IFN-γ-producing CD4⁺ T cells was notsignificantly affected by any of the re-colonization conditions (FIG.7E). Next, we generated a B. theta mutant lacking the β-gal BT1626 geneencoding for the MYH6 peptide mimic (B. theta β-gal) (FIG. 7F).Mono-colonization of GF TCRM mice with B. theta β-gal or the parentalstrain (FIG. 2I) resulted in efficient seeding of the intestinalmicroenvironments with both strains (FIG. 7G), but precipitatedsignificantly reduced accumulation of CD45⁺ immune cells and Vβ8⁺CD4⁺ Tcells in the myocardium (FIG. 2J). Moreover, IL-17 production ofmyosin-specific CD4⁺ T cells in heart and colon was significantlyreduced when TCRM mice were re-colonized with B. theta β-gal (FIG. 2K).Assessment of IgA binding to intestinal bacteria using bacterial flowcytometry (FIG. 7H) indicated that the presence of TCRM cellssubstantially increases the production of IgA against commensalmicrobiota under SPF conditions (FIG. 7I). To determine to which extentsuch increased intestinal IgA production depends on specific antigenicstimulation by the MYH6 mimic peptide, we assessed B. theta-specific IgAbinding in GF TCRM mice that were mono-colonized with B. theta β-gal.Indeed, the absence of the cross-reactive epitope in B. theta β-gal ledto a significantly reduced IgA response when compared to re-colonizationwith the parental B. theta strain (FIG. 2L and M). In sum, these datareveal that organ-specific, genuinely autoreactive CD4⁺ T cells canshape the colonic microbiome and that distinct bacterial communities,here Bacteroides, represent a source of mimic peptides that can activateMYH6-specific CD4⁺ T cells.

Antibiotics Treatment Mitigates Inflammatory Cardiomyopathy

Since the microbiome can be manipulated through the utilization ofantibiotics, we assessed whether reduction of Bacteroides communities bybroad-spectrum antibiotics combination (Sulfadoxine, Trimethoprim andMetronidazol; S+T+M) affects disease progression in TCRM mice. We foundthat antibiotics treatment resulted in a substantial shift in themicrobiome composition including the reduced abundance of Bacteroides(FIG. 8A). None of the treated TCRM mice succumbed to lethalinflammatory cardiomyopathy (FIG. 3A), which was most likely due tosignificantly reduced cardiac inflammation under antibiotic treatment(FIG. 3B and C). To corroborate these findings and to dissect theactivation patterns of cardiac myosin-specific T cells, we utilized theadoptive transfer model of TCRM splenocytes into Rag1^(−/−) mice withantibiotics treatment starting one week before the splenocyte transfer(FIG. 3D). As expected, treatment with broad-spectrum antibiotics suchas Vancomycin reduced not only B. theta levels in fecal samples (FIG.3E), but significantly ameliorated the cardiac disease at day 28 postadoptive transfer (FIG. 3F). Flow cytometric analysis confirmed theprofound reduction of the cardiac inflammation (FIG. 3G) withsignificantly reduced accumulation of TCR-transgenic CD4⁺ T cells inhearts of Rag1^(−/−) recipients treated with Streptomycin, S+T+M orVancomycin (FIG. 3H). Likewise, these three antibiotic regimens reducedCD45⁺ immune cell (FIG. 8B) and Vβ8⁺ CD4⁺ T cell accumulation in thecolon (FIG. 8C), but not in the spleen of Rag1^(−/−) recipients (FIG.8D). Vancomycin and S+T+M antibiotics treatment had a pronounced impacton the activation of heart-infiltrating and colonic Th17 cells, whilethe activity of Th1 cells was reduced to a lesser extent (FIG. 3I andFIG. 8E-F). The adoptively transferred B cells from TCRM spleensgenerated B. theta-specific IgG antibodies that could be detected in theserum of Rag1^(−/−) recipients on day 28 post transfer (FIG. 3J).Antibiotics treatment reduced B. theta-specific IgG antibodies, but hadvariable effects on IgG antibody responses against B. distasonis, B.vulgatus or Enterobacter cloacae (FIG. 3J) indicating that antibioticstreatment did not negatively impact global immune reactivity in theRag1^(−/−) recipients. These results and the finding that adoptivetransfer of heart-specific TCRM cells to BALB/c mice significantlyenhanced anti-B. theta antibody responses (FIG. 3K) further support thenotion that heart-specific CD4⁺ T cells specifically interact withmicrobial components in the intestine and thereby impact systemic immunereactivity.

T and B Cell Reactivity Against Bacteroides in Human MyocarditisPatients

To establish translational relevance of the link between microbialmolecular mimicry and inflammatory cardiomyopathy, we assessedanti-Bacteroides IgG responses in sera from patients with endomyocardialbiopsy-proven myocarditis (Etics/AMITIS study, Table 2) (27). Patientswith acute myocarditis showed significantly elevated B. theta-specificIgG responses compared to a healthy control group (FIG. 4A). Clinicalimprovement of the myocarditis patients with gain of heart function(FIG. 4B) and reduced inflammatory status (FIG. 4C) was accompanied by areduction in seroreactivity against B. theta (FIG. 4A). Based on anti-B.theta IgG reactivity in patients with acute myocarditis, we grouped thepatients into high (>mean, red box in FIG. 4A) and low (<mean, black boxin FIG. 4A) responders and correlated their antibody status with theavailable clinical parameters: positivity for anti-beta 1 adrenergicreceptor autoantibodies (FIG. 9A), C-reactive protein (CRP) levels (FIG.9B) and an ejection fraction cut-off of 40% (FIG. 9C). Acute myocarditispatients with low anti-B. theta reactivity showed a combined clinicalscore that was significantly lower compared to the high responder group(FIG. 4D) indicating that systemic anti-Bacteroides immune reactivity islinked to myocardial inflammation in humans.

While IgG antibodies against B. theta in the AMITIS cohort werediminished at subsequent visits (FIG. 4A), anti-Enterobacter cloacae(FIG. 9D), anti-Escherichia coli antibodies (FIG. 9E) and total serumIgG concentrations (FIG. 9F) did not change significantly. Moreover, IgGantibody reactivity against other Bacteroides species (B. distasonis andB. vulgatus) was not different in AMITIS myocarditis patients andhealthy controls (FIG. 4E). To corroborate these results, we establisheda prospective clinical study and included thirteen acute myocarditispatients showing typical late enhancement pattern in cardiac magneticresonance imaging and four acute heart failure patients with leftventricular ejection fraction ≤40% (Micro-DCM study; Table 3). A cohortof age-matched healthy volunteers served as controls (Table 4).Importantly, myocarditis patients of the Micro-DCM study showedsignificantly elevated anti-B. theta IgG antibodies (FIG. 4F), whileseroreactivity against other bacteria—except reduced reactivity againstB. vulgatus—were not different when compared to healthy controls (FIG.4G).

Preclinical studies in mice have established that MYH6 is the dominantautoantigen in the heart tissue (5, 28) and that susceptibility toexperimental autoimmune myocarditis depends mainly on the MHC haplotype,whereby MYH6 epitopes appear to be presented by different MHC class IImolecules (29). Bioinformatics analysis of binding affinities of the B.theta β-gal₁₁₋₂₅ peptide mimic indicated several HLA-DQA1*/HLA-DQB1*combinations, including HLA-DQ8, that are also predicted to bind humanMYH6₆₁₄₋₆₂₉ (FIG. 4H). Assessment of T cell reactivity against the MYH6and β-gal peptides in peripheral blood mononuclear cells Micro-DCMpatients revealed that patients exhibit significantly higher IFN-

reactivity against both peptides when compared to healthy controls (FIG.4I). Moreover, the frequency of IFN-

-producing T cells in Micro-DCM patients that possess alleles other thanHLA-DQB1*03 was also reduced in comparison to the HLA-DQB1*03-positivegroup (FIG. 4I). When we focused the analysis on Micro-DCM myocarditispatients and individuals showing a T cell response >0 in the ELISpotassay for at least one of the peptides, we found a highly significantcorrelation between MYH6 and β-gal₁₁₋₂₅ peptide reactivity (FIG. 9K)suggesting that heart-specific CD4⁺ T cells cross-react with thebacterial peptide in human myocarditis patients. Finally, to provideevidence that the presence of B. theta in the human intestinalmicrobiome is causally linked to myocarditis development, we performedfecal transplants from B. theta-positive myocarditis patients into GFTCRM and transgene-negative mice (FIG. 4J). Both groups of mice wereequally well colonized by B. theta (FIG. 4K). Increased accumulation ofCD45⁺ immune cells (FIG. 4L) and CD4⁺ T cells (FIG. 4M) in TCRM heartsafter four weeks of re-colonization indicates that the microbiome ofhuman myocarditis patients contains the pathological principle thatdrives myocardial inflammation in TCRM mice. In sum, the causal evidenceprovided by the preclinical studies in TCRM mice and the significantcorrelation of systemic anti-Bacteroides immune reactivity with diseaseseverity in human myocarditis patients indicate that inflammatorycardiomyopathy in humans is driven—at least partially—through theactivation of heart-specific Th cells by bacterial peptide mimics thatare derived from the intestinal microbiota.

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We claim:
 1. A method of preventing myocarditis in a subject in needthereof, comprising reducing the amount of Bacteroides sp. in a subject.2. A method of treating myocarditis or DCM in a subject in need thereof,comprising reducing the amount of Bacteroides sp. in a subject.
 3. Amethod of limiting progression of myocarditis toward DCM in a subject inneed thereof, comprising reducing the amount of Bacteroides sp. in asubject.
 4. A method of diagnosis a subject as having myocarditis orDCM, comprising obtaining a biological sample of the subject,quantifying the amount of Bacteroides sp. in the biological samplerelative to a control sample.
 5. The method of claim 1, comprisingadministering to the subject an effective amount of an antibiotic,phage, recombinant phage, packaged phagemid, bacteria, engineeredbacteria, bacteriocin or endolysin.
 6. The method of claim 2, comprisingadministering to the subject an effective amount of an antibiotic,phage, recombinant phage, packaged phagemid, bacteria, engineeredbacteria, bacteriocin or endolysin.
 7. The method of claim 3, comprisingadministering to the subject an effective amount of an antibiotic,phage, recombinant phage, packaged phagemid, bacteria, engineeredbacteria, bacteriocin or endolysin.
 8. The method of claim 4, comprisingadministering to the subject an effective amount of an antibiotic,phage, recombinant phage, packaged phagemid, bacteria, engineeredbacteria, bacteriocin or endolysin.
 9. The method of claim 5, whereinthe antibiotic is selected from streptomycin, vancomycin, clindamycin,metronidazole, sulphadoxine, trimethoprim, or any combination of 1, 2,3, 4, 5 or 6 of these antibiotics.
 10. The method of claim 6, whereinthe antibiotic is selected from streptomycin, vancomycin, clindamycin,metronidazole, sulphadoxine, trimethoprim, or any combination of 1, 2,3, 4, 5 or 6 of these antibiotics.
 11. The method of claim 7, whereinthe antibiotic is selected from streptomycin, vancomycin, clindamycin,metronidazole, sulphadoxine, trimethoprim, or any combination of 1, 2,3, 4, 5 or 6 of these antibiotics.
 12. The method of claim 8, whereinthe antibiotic is selected from streptomycin, vancomycin, clindamycin,metronidazole, sulphadoxine, trimethoprim, or any combination of 1, 2,3, 4, 5 or 6 of these antibiotics.
 13. The method of claim 5, whereinthe phage, recombinant phage or packaged phagemid encodes a nucleaseselected from CRISPR-Cas, TALENs and variants, zinc finger nuclease(ZFN) and ZFN variants, natural, evolved or engineered meganuclease orrecombinase variants.
 14. The method of claim 6, wherein the phage,recombinant phage or packaged phagemid encodes a nuclease selected fromCRISPR-Cas, TALENs and variants, zinc finger nuclease (ZFN) and ZFNvariants, natural, evolved or engineered meganuclease or recombinasevariants.
 15. The method of claim 7, wherein the phage, recombinantphage or packaged phagemid encodes a nuclease selected from CRISPR-Cas,TALENs and variants, zinc finger nuclease (ZFN) and ZFN variants,natural, evolved or engineered meganuclease or recombinase variants. 16.The method of claim 8, wherein the phage, recombinant phage or packagedphagemid encodes a nuclease selected from CRISPR-Cas, TALENs andvariants, zinc finger nuclease (ZFN) and ZFN variants, natural, evolvedor engineered meganuclease or recombinase variants.
 17. The method ofclaim 5, wherein the Bacteroides is B. thetaiotaomicron and/or B.faecis.
 18. The method of claim 6, wherein the Bacteroides is B.thetaiotaomicron and/or B. faecis.
 19. The method of claim 7, whereinthe Bacteroides is B. thetaiotaomicron and/or B. faecis.
 20. The methodof claim 8, wherein the Bacteroides is B. thetaiotaomicron and/or B.faecis.